Method for protein nanowire synthesis and tunable control of nanowire length

ABSTRACT

Methods for synthesizing nanowires are provided. Modified PilA peptides are used as peptide building blocks for synthesizing the nanowires. The method places the peptide building blocks in an assembly buffer with a hydrophobe. Addition of a hydrophobe and molecular crowding by evaporation of the assembly buffer triggers the self-assembly of the peptide building blocks into fibers. Multiple elongation cycles of addition of peptide building blocks, mixing and evaporation are conducted to promote elongation of the fibers and synthesis of nanowires. Electronic characterization of the synthesized nanowires is provided.

The present application claims benefit under 35 U.S.C. 119(e) of U.S.Provisional Application Ser. No. 62/896,179, filed on Sep. 5, 2019.

STATEMENT OF GOVERNMENT RIGHTS

This invention was made with government support under MCB1021948 and1629439 awarded by the National Science Foundation, and under ES017052awarded by the National Institutes of Health. The government has certainrights in the invention.

BACKGROUND

Semiconductor electronics have exhibited a sustained exponentialdecrease in size and cost with a similar increase in performance overthe last thirty years. While such progress is expected to continue, theeconomics and/or physical barriers of continued use of silicon forincreasingly small and more powerful devices will ultimately pose achallenge. Moreover, increases in pollution have been tied withincreased energy consumption for at least the last several hundredyears. Accelerated global warming and environmental degradation make thedevelopment of alternative energy sources an urgent priority. The worldtherefore needs new sources of energy and new materials for use in fuelcells and nanoelectronic devices.

SUMMARY

Microbes have the potential to address the problems of pollution, theneed for clean affordable energy and the need for new nanoelectronicmaterials. The present description relates to synthesis of microbialnanowires that conduct electricity. Such nanowires are made frompeptides inspired in microbial pilins such as modified PilA peptides,which are the structural subunit of microbial protein appendages knownas pili. In one embodiment, the description herein relates to expressionof modified PilA peptides in a recombinant system and to the formationof functional pilin nanowires using the modified PilA peptides. In someembodiments, the nucleic acids encoding the pilin peptides arerecombinantly modified, expressed and isolated. The isolated PilApeptides can then be assembled to form conductive pilin nanowires. Inone embodiment, the modified PilA peptides are synthesized PilApeptides. The ability for modified PilA peptides, such as truncated PilApeptides, to form functional nanowires can enable production of largequantities of conductive pilin nanowires.

In one aspect, the present description relates to methods forsynthesizing protein nanowires from modified PilA peptides. The modifiedPilA peptides may be recombinant PilA peptides. The modified PilApeptides may be truncated PilA peptides. The truncated PilA peptides caninclude peptides comprising an amino acid sequence selected from SEQ IDNO:1-5. In some embodiments, the amino acid sequence of the disclosedmodified PilA peptides may be genetically or chemically modified so thenanowires formed from the modified PilA peptide has electricalconductivity or other desirable activity. In other embodiments, thenanowires formed from the modified PilA peptides can have modifiedadhesive or coupling properties relative to naturally occurring nanowirepolypeptides.

Another aspect of the invention is a pilus or pili that includes such amodified nanowire polypeptide. Further aspects of the invention use thedisclosed pilin nanowires in devices and for soluble metal remediation.

In one embodiment, the present description includes a method ofsynthesizing protein nanowires. The method can include providingpurified peptide building blocks. In one embodiment, the peptidebuilding blocks may be isolated from a recombinant host. The method caninclude suspending the purified peptide building blocks in an assemblybuffer. The method can include forming an assembly composition by addinga hydrophobe to the assembly buffer and peptide building blocks totrigger self-assembly of the peptide building blocks. The method caninclude increasing molecular crowding by evaporation of a volume of theassembly buffer in the assembly composition to facilitate hydrophobeguided assembly of conductive nanowires.

In one embodiment, the method can further comprise conducting one ormore elongation cycles to promote fiber formation. In one embodiment,the elongation cycle can include providing additional peptide buildingblocks by refeeding the assembly composition. In one embodiment, theelongation cycle may include providing additional hydrophobe into theassembly composition. In one embodiment, the elongation cycle can alsoinclude mixing the assembly composition and evaporating the assemblybuffer from the assembly composition.

In one embodiment, the method can comprise conducting about 4 cycles ofelongation. In one embodiment, the hydrophobe is octadecane. In oneembodiment the octadecane is provided in surface-constrained form.

In one embodiment, the hydrophobe is added to the assembly compositionby loading the peptide building blocks into a column comprisingparticles and eluting the peptide building blocks into the assemblybuffer, wherein the elution results in the co-elution of a portion ofthe particles of the column and the peptide building blocks into theassembly buffer and wherein the particles of the column are thehydrophobes in the assembly composition. In one embodiment, thehydrophobe is C18-silica particles.

In one embodiment, the peptide building blocks are recombinant modifiedPilA peptides. In one embodiment, the recombinant host is E. coli. Inone embodiment, the peptide building blocks are truncated PilA peptidesfrom G. sulfurreducens. In one embodiment, the peptide building blocksare PilA₁₉ peptides from G. sulfurreducens. In one embodiment, thepeptide building blocks are truncated PilA_(n) peptides, wherein thetruncated PilA_(n) peptides have the amino acid sequences selected fromthe group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQID NO:5.

In one embodiment, the nanowires formed have a length from about 0.5 μmto about 10 μm. In some embodiments, the nanowires may have a lengthfrom about 2 μm to about 8 μm or from about 4 μm to about 7 μm, furtherincluding any range therebetween.

In one embodiment, the present description includes a compositioncomprising synthesized protein nanowires. The protein nanowires may berecombinant protein nanowires. In one embodiment, the nanowires caninclude peptide building blocks. The peptide building blocks may bederived from PilA peptide. The peptide building blocks may be modifiedPilA peptides. The peptide building blocks may be recombinant peptidebuilding blocks. In one embodiment, the nanowires have a length of fromabout 1 μm to about 10 μm.

In one embodiment, the peptide building blocks are truncated PilApeptides. In one embodiment, the peptide building blocks are PilA₁₉peptides. In one embodiment, the nanowires have a length from about 0.5μm to about 10 μm or from about 5 μm to about 7 μm, further includingany range therebetween.

In one embodiment, the nanowires have an average diameter of from about1 nm to about 3 nm. In one embodiment, the nanowires have an averagediameter of about 2 nm. In one embodiment, the nanowires have a CircularDichroism (CD) profile with a 222 nm/208 nm intensity ratio of fromabout 0.7 to about 0.9. In one embodiment, the nanowires have a CDprofile with a 222 nm/208 nm intensity ratio from about 0.9 to about 2.In one embodiment, the nanowires have a CD profile with a 222 nm/208 nmintensity ratio about 2 or higher.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1A is a model diagram of the molecular structure of a Geobactersulfurreducens (Gsu) pilus fiber optimized via molecular dynamics (MD)showing aromatic residues (phenylalanines in green, tyrosines in orange)and charged amino acids involved in salt bridges (acidic in red, basicin blue).

FIG. 1B is a diagram of the alignment of the amino sequence of the PilAmonomer and the N-terminus truncated PilA_(n) derivatives.

FIG. 1C is a graph that shows Kyte-Doolittle (black) and AGGRESCAN (red)plots and analyses of the PilA pilin with signal peptide (prepilin) andcut-off values (horizontal lines) of hydrophobicity and aggregationpotential scores.

FIG. 1D is a model via MD of the mature PilA pilin and PilA derivativeshighlighting aromatic and charge amino acids in FIG. 1A.

FIG. 2 is a photograph of the effect of temperature and incubation timewith DTT inducer on cleavage of the PilA_(n) peptides from the CBDmodule. The full fusion (CBD-PilA_(n)) and peptide-free CBD proteinsbound to the chitin beads were solubilized by boiling with 1% SDS andseparated in a 7.5% glycine SDS-PAGE. The approximate migration of thetwo bands is indicated in reference to molecular mass standards (numbersat left). Lanes: no DTT (−) or incubation with DTT (+) for 24, 48 or 72h. The ratio of the lower band (CBD) over the slow migrating band(CBD-PilA_(n)) serves as a proxy of cleavage efficiency.

FIG. 3 is a plot of MALDI-TOF MS analysis of PilAn peptides eluted fromchitin column after cleavage with DTT for 24 h at 23° C. The theoreticalmolecular mass of the truncated pilins is: 5,431 Da (PilA₁₀), 4,595 Da(PilA₁₉), 4,524 Da (PilA₂₀), and 4,314 Da (PilA₂₂).

FIG. 4A is a photograph of SDS-PAGE gel showing the enrichment of theCBD-PilA₁₉ fusion protein (black arrow) in the soluble (lanes 1-2)rather than insoluble (lane 3-4) proteins from two independent culturelysates. Lane 5 shows the migration of the recombinant PilA₁₉ peptide(red arrow) eluted from the chitin column after DTT-induced cleavagefrom the CBD domain. Numbers at right are molecular weight standards inkDa.

FIG. 4B is a plot of the effect of SDS detergent on PilA₁₉ helicity byCircular Dichroism (CD). The plot shows the CD spectra collected atincreasing concentrations of SDS at pH 7.

FIG. 4C is a plot of the effect of pH on molar ellipticities at keywavelengths in the CD spectra for pH 3.8 and pH 7.

FIG. 5A is a schematic diagram of evaporation-induced self-assembly ofrecombinant PilA₁₉ peptides. The protocol illustrates the key steps inthe assembly of PilA₁₉ fibers. The evaporation-induced assembly includedsequential additions of the peptide solution to elongate the fibers.

FIG. 5B is a plot (top) and an image (bottom) showing the hydrophobe(C18-silica particles) dose effect on fiber formation, estimated asaverage fiber size by dynamic light scattering (plot, on top) and AFMimaging on HOPG (bottom; images are for 0.2 or 3.5 mM C18-silicareaction mixes only).

FIG. 5C is a plot (top) and an image (bottom) showing the effect ofreaction mixing on fiber elongation (box plot). Boxes in plot contain50% of all values and whiskers represent the 25th and 75th percentilesof fiber lengths measured by analyzing AFM images of random fields withsamples (bottom) with the ImageJ software. The median is shown as ahorizontal line across the boxes, average as a cross, and outliers ascircles outside the boxes. Scale bar in the AFM images in FIGS. 5B and5C is 1 m.

FIG. 6A is plot of the UV-Vis spectra showing the effect of C18-columnwash on nucleator concentration. UV-vis spectra of eluants fromtriplicate C18-columns washed with 5 ml (dark color; optimal protocolfor PilA₁₉ fiber formation) or 18 ml (light color; extended washpreventing PilA₁₉ fiber formation) of ddH₂O prior to buffer exchangewith assembly buffer. Reduced washes produce the spectral peaks for DTT(˜205 nm) and silica (˜245 nm). The silica peak is produced byC18-silica particles 25-50 nm in diameter (AFM image in inset).

FIG. 6B are images showing the effect of C18-silica hydrophobe on PilA₁₉fiber formation. Tapping mode AFM showing optimal fiber formation in thepresence (top) but not in the absence (bottom) of the C18-silicaparticles.

FIG. 6C is a plot of standard curves of absorbance at 245 nm of silicain solutions made with assembly buffer and the column's C18-silicaresin.

FIG. 6D is a plot of standard curves of absorbance at 245 nm of silicain solutions made with assembly buffer and pure silica particles ofsizes similar to those of the column's resin (63-200 m) (FIG. 6C) andeluted particles (0.5-1 m).

FIGS. 7A-7F are images showing the effect of C18-column pre-wash. FIGS.7A-7B are images of C18-columns washed with 5 ml (FIG. 7A), 15 ml (FIG.7B) or 30 ml (FIG. 7C) of ddH₂O on PilA₁₉ fiber formation to reduce theamount of loosely-bound C15-silica particles co-eluting with PilA₁₉during the buffer column exchange step. FIGS. 7D-7F are images of PilA₁₉fiber formation of a buffer-only control. The eluents were subjected tothe evaporation induced assembly protocol and samples deposited on HOPGwere imaged with an AFM operated in tapping mode.

FIG. 8A is an image of AFM imaging of PilA₁₉ fibers assembled in vitro.Inset shows single fibers and braided supramolecular structures withline scans used to determine the AFM height (red, single fiber; blue,braided fibers). Scale, 0.2 μm.

FIG. 8B is plot of CD spectra of recombinant pili.

FIG. 8C is a plot of CD spectra of native pili treated with Urea (8Murea; dash) or untreated (solid line).

FIG. 9A is a plot of induction of PilA₁₉ fiber formation withoctadecane. The plot is UV-vis spectrum of eluent collected from anOasis Max™ exchange cartridge after a buffer exchange showing thepresence of DTT (205 nm, inset shows standard curve).

FIG. 9B is an image of a tapping mode AFM showing PilA₁₉ fiber formationin the presence of octadecane (1:100 aqueous solution) when using OasisMax™ Cartridges for peptide buffer exchange. Controls without PilA₁₉show the resin residues and an octadecane layer on the electrodesurface. Scale bar, 1 m.

FIGS. 10A-10H are images and plots of electronic characterization ofrecombinant pili. FIGS. 10A and 10B are AFM amplitude images ofrecombinant (FIG. 10A) and native (FIG. 10B) pili on HOPG (scale bars,200 nm) with FIG. 10C and FIG. 10D showing representative I-V curves oftheir CP-AFM transversal conductivity (average, in black). FIGS. 10E and10F are room temperature STM topographic images of untreated (FIG. 10E)or chemically-fixed (FIG. 10F) recombinant pili (0.5 V, 350 picoampere(pA); scale bar, 100 nm). FIGS. 10G-10H are average I-V tunnelingspectra of 2 sequential measurements for each of two pilus regions inuntreated (black) and fixed (green) samples (FIG. 10G). FIG. 10H showsdifferential conductance (dI/dV) curves of the untreated and chemicallytreated pili, calculated as the numerical derivative of the I-V curvesshown in FIG. 10G.

FIG. 11 is a schematic diagram of steps and model of PilA₁₉ fiberformation. (Left) Steps in the bottom-up fabrication of proteinnanowires with PilA₁₉ peptide building blocks triggered in the presenceof a hydrophobe. (Right) Nucleation-dependent polymerization model ofhydrophobe-triggered pilin assembly showing the three phases ofnucleation, fiber elongation, and saturation.

DESCRIPTION OF THE EMBODIMENTS

In the following detailed description, the embodiments are described insufficient detail to enable those skilled in the art to practice them,and it is to be understood that other embodiments may be utilized andthat chemical and procedural changes may be made without departing fromthe spirit and scope of the present subject matter. The followingdetailed description is, therefore, not to be taken in a limiting sense,and the scope of the embodiments is defined only by the appended claims.

The embodiments described herein can include a novel method forsynthesizing protein nanowires. The protein nanowires may be synthesizedusing recombinant peptide building blocks. In one embodiment, thepeptide building blocks can be derived from the conductive pilin peptideof the electrically active bacterium Geobacter sulfurreducens. Thesynthesis of nanowires can include nucleation and elongation steps forefficient peptide self-assembly and control of nanowire length. Themethod can include addition of a hydrophobe. The hydrophobe may be, forexample, octadecane, provided in aqueous solution. The hydrophobe mayalso be materials such as functionalized silica beads. The hydrophobecan trigger the self-assembly of the peptides and fiber formation. Themethod may also include increasing molecular crowding via evaporation tofacilitate hydrophobe-guided nucleation and assembly of the peptidesinto conductive protein fibers. Additional refeeding steps with mixingmay be used to control the length of the nanowires and the yields ofnanowires. The method can yield protein nanowires that retain thebiochemical and electronic properties of the native protein nanowires(pili) produced by Geobacter cells even under chemical fixation, acritical consideration for integration in electronic devices.

Unlike the native protein nanowires, synthesized protein nanowires canbe mass-produced in a scalable process. This process may use arecombinant host (e.g., E. coli) to produce the genetically engineeredpeptide building blocks and can rely on molecular crowding in thepresence of a hydrophobe to induce peptide self-assembly into proteinnanowires of defined length. This can result in design and massproduction of generations of protein nanowires for custom applications.For example, if only currently available technologies were employed, thecosts of building the necessary manufacturing facilities will becomeprohibitive due to the shrinking size of devices, heat dissipationproblems due to closely packed structures, non-uniformity in dopant andconductive materials, and high electric fields that may lead to acascade of breakdown events within closely packed components.

Various terms are defined herein. See also definitions in U.S. Pat. Nos.9,716,287 and 10,074,867, both of which are incorporated herein byreference. In case of a conflict in the meaning of various terms, thedefinitions provided herein prevail.

The terms “preferred” and “preferably”, “example” and “exemplary” referto embodiments that may afford certain benefits, under certaincircumstances. However, other embodiments may also be preferred orexemplary, under the same or other circumstances. Furthermore, therecitation of one or more preferred or exemplary embodiments does notimply that other embodiments are not useful and is not intended toexclude other embodiments from the inventive scope of the presentdisclosure.

The singular forms of the terms “a”, “an”, and “the” as used hereininclude plural references unless the context clearly dictates otherwise.For example, the term “a tip” includes a plurality of tips.

Reference to “a” chemical compound refers one or more molecules of thechemical compound, rather than being limited to a single molecule of thechemical compound. Furthermore, the one or more molecules may or may notbe identical, so long as they fall under the category of the chemicalcompound.

The terms “at least one” and “one or more of” an element are usedinterchangeably and have the same meaning that includes a single elementand a plurality of the elements, and may also be represented by thesuffix “(s)” at the end of the element.

The terms “about” and “substantially” are used herein with respect tomeasurable values and ranges due to expected variations known to thoseskilled in the art (e.g., limitations and variability in measurements).

The terms “and/or” means one or all of the listed elements or acombination of any two or more of the listed elements.

The terms “comprises,” “comprising,” and variations thereof are to beconstrued as open ended—i.e., additional elements or steps are optionaland may or may not be present.

The term “polypeptide” as used herein refers to at least two amino acidresidues connected as a chain via covalent bonds such as peptide bonds,and can be recombinant polypeptides, natural polypeptides or syntheticpolypeptides. The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein

The terms “recombinant polypeptide” and “recombinant peptide” as usedherein refers to a manipulated form of a polypeptides encoded byrecombinant DNA which has been cloned in a foreign expression system tosupport the expression of the exogenous gene.

The term “peptide building blocks” as used herein refers to peptidesthat serve as the units for assembly into larger protein complexes suchas fibers and/or nanowires. The peptide building blocks can berecombinant peptides and/or synthesized peptides. In one exemplaryembodiment, the peptide building blocks are modified PilA peptides.

The term “fibers” as used herein refers to complexes of peptide buildingblocks that associate to form filamentous supramolecular proteinstructures. Fibers may or may not transport charges.

The term “nanowires” as used herein refers to complexes of pilinpeptides that form conductive fiber assemblies. Nanowires havefiber-like geometry and transport charges.

The term “synthesized nanowire” as used herein refers to nanowiresformed by purified pilin peptide(s). The pilin peptides may besynthesized such as by chemical methods. The pilin peptides may besynthesized by recombinant methods. The pilin peptides may be encoded byrecombinant DNA which has been cloned in a foreign expression system tosupport the expression of the exogenous gene and form recombinantpeptides. Synthesized nanowires in the present description may havemodified pilin peptides relative to naturally occurring nanowires.

The term “recombinant nanowire” as used herein refers to nanowiresformed by pilin peptide(s) encoded by recombinant DNA which has beencloned in a foreign expression system to support the expression of theexogenous gene. Recombinant nanowires in the present description mayhave modified pilin peptides relative to naturally occurring nanowires.

The term “genetically engineered” as used herein refers to a manipulatedform of a polypeptide or a peptide encoded by recombinant DNA which hasbeen cloned in a foreign expression system to support the expression ofthe exogenous gene.

The term “pilin peptides” or “PilA peptides” as used herein refers toPilA peptides. The term “pilin peptides” as used herein includesmodified PilA peptides derived from the naturally occurring PilApeptides.

The term “derived” as used herein refers to modified peptides or genesrelative to the naturally occurring peptide or gene. A modified orgenetically engineered PilA peptide derived from naturally occurringPilA peptide has from about 10% identity to about 99% identity to thenaturally occurring PilA peptide, or at least from about 10% identity toabout 99% identity to the naturally occurring identity. The modifiedPilA peptide can keep the helical structure and assembly. Theconductivity of the modified PilA peptide can depend on the presence ofthe aromatic and charged residues and formation of salt bridges betweenneighboring peptides.

The term “modified PilA peptides” as used herein refers to pilinpeptides that have one or more amino acids that are different comparedto the naturally occurring PilA polypeptide. Modified PilA peptides mayhave one more amino acids that are substituted and/or deleted ortruncated compared to the naturally occurring PilA polypeptide.

The term “truncated PilA peptides” or “PilA_(n)” as used herein refersto pilin peptides that have one or more amino acids deleted compared tothe naturally occurring PilA polypeptide. Examples of truncated PilApeptides can include PilA₁₀, PilA₁₉, PilA₂₀, PilA₂₂ and the like.

The term “PilA₁₉” as used herein refers to a PilA peptide that has thefirst 19 amino acids from the N-terminus of the PilA polypeptidedeleted.

The term “pilus” (singular) or “pili” (plural) as used herein refers tocomplexes formed by pilin peptides and are electrically conductive.

The term “protein nanowires” as used herein refers to the complex ofpolypeptides formed into filaments that are electrically conductive.

The term “pilus nanowires” as used herein refers to filaments that areelectrically conductive and formed from PilA peptides, modified PilApeptides and/or truncated PilA peptides.

The term “assembly buffer” as used herein refers to a buffer thatmaintains peptide building blocks such as pilin peptides and modifiedpilin peptides in solution.

The term “assembly composition” as used herein refers to a compositionthat includes assembly buffer, peptide building blocks and a hydrophobeand can initiate self-assembly of the peptide building blocks intofibers.

The term “hydrophobe” as used herein refers to a molecule and/ormaterial that is hydrophobic in nature and can serve as the nucleationsite for the self-assembly of the peptide building blocks. Hydrophobescan be materials that can be coated with hydrophobic molecules togenerate materials with hydrophobic characteristics.

The term “bottom-up fabrication” as used herein refers to synthesizedprotein nanowires.

The term “tunable control” as used herein refers to the ability toadjust experimental parameters to modify the characteristics of thematerial.

The term “substantial identity” as used herein refers to a peptide,protein or nucleic acid comprises a sequence with from about 10 to about100% sequence identity to a reference sequence.

The term “biofilm” as used herein refers to a community of microbesparticularly bacteria attached to a surface with the community membersbeing contained or protected by a self-generated extracellular polymericmatrix or EPS.

The term “substrate” as used herein refers to a substance to whichanother substance binds or connects.

The term “conductance” refers to a material property whereby electronsmigrate through the material in response to an applied voltage(difference in electrical potential) across the material. The rate ofelectron migration (charge/time) is the electrical current passingthrough the material. Materials that exhibit conductance are referred toas conductors.

In general, amino acids can be placed into three main classes:hydrophilic amino acids, hydrophobic amino acids and cysteine-like aminoacids, depending primarily on the characteristics of the amino acid sidechain. These main classes may be further divided into subclasses. Forexample, some types of hydrophobic amino acids have aromatic side chainswhile other types of hydrophobic amino acids do not have aromatic sidechains. Moreover, aromatic amino acids can have functional groups thatprovide a more hydrophilic character and that permit acceptance andtransport of electrons (e.g., tyrosine). In general, the hydrophilicand/or aromatic amino acids have a more direct role in the electricalconductivity functions of the pilus nanowires.

Hydrophilic amino acids include amino acids having acidic, basic oruncharged polar side chains and hydrophobic amino acids include aminoacids having a polar side chains. A polar amino acids may be furthersubdivided to include, among others, aliphatic amino acids. Thedefinitions of the classes of amino acids as used herein are as follows:

The term “hydrophobic amino acid” as used herein refers to an amino acidhaving a side chain that is uncharged at physiological pH and that isrepelled by aqueous solution. Examples of genetically encodedhydrophobic amino acids include Ala, Ile, Leu and Val.

The term “aromatic amino acid” as used herein refers to a hydrophobic orhydrophilic amino acid having a side chain containing at least one ringhaving a conjugated r-electron system (aromatic group). The aromaticgroup may be further substituted with substituent groups such as alkyl,alkenyl, alkynyl, hydroxyl, sulfonyl, nitro and amino groups, as well asothers. Examples of genetically encoded aromatic amino acids includephenylalanine, tyrosine and tryptophan. Commonly encounterednon-genetically encoded aromatic amino acids include phenylglycine,2-naphthylalanine, β-2-thienylalanine, 1,2,3,4tetrahydroisoquinoline-3-carboxylic acid, 4-chlorophenylalanine,2-fluorophenylalanine, 3-fluorophenylalanine and 4-fluorophenylalanine.

The term “a polar amino acid” as used herein refers to a hydrophobicamino acid having a side chain that is generally uncharged atphysiological pH and that is not polar. Examples of genetically encodeda polar amino acids include proline and methionine. Examples ofnon-encoded a polar amino acids include Cha.

The term “aliphatic amino acid” as used herein refers to an apolar aminoacid having a saturated or unsaturated straight chain, branched orcyclic hydrocarbon side chain. Examples of genetically encoded aliphaticamino acids include Ala, Leu, Val and Ile.

The term “hydrophilic amino acid” as used herein refers to an amino acidhaving a side chain that is attracted by aqueous solution. Examples ofgenetically encoded hydrophilic amino acids include Ser and Lys.

The term “acidic amino acid” or “negatively charged amino acid” as usedherein refers to a hydrophilic amino acid having a side chain pK valueof less than 7. Acidic amino acids typically have negatively chargedside chains at physiological pH due to loss of a hydrogen ion. Examplesof genetically encoded acidic amino acids include aspartic acid(aspartate) and glutamic acid (glutamate).

The term “basic amino acid” as used herein refers to a hydrophilic aminoacid having a side chain pK value of greater than 7. Basic amino acidstypically have positively charged side chains at physiological pH due toassociation with hydronium ion. Examples of genetically encoded basicamino acids include arginine, lysine and histidine. Examples ofnon-genetically encoded basic amino acids include the non-cyclic aminoacids ornithine, 2,3-diaminopropionic acid, 2,4-diaminobutyric acid andhomoarginine.

The term “polar amino acid” as used herein refers to a hydrophilic aminoacid having a side chain that is uncharged at physiological pH, butwhere a bond in the side chain has a pair of electrons that are heldmore closely by one of the atoms involved in the bond. Examples ofgenetically encoded polar amino acids include asparagine and glutamine.Examples of non-genetically encoded polar amino acids includecitrulline, N-acetyl lysine and methionine sulfoxide.

The term “cysteine-like amino acid” as used herein refers to an aminoacid having a side chain capable of forming a covalent linkage with aside chain of another amino acid residue, such as a disulfide linkage.Typically, cysteine-like amino acids generally have a side chaincontaining at least one thiol (SH) group. An example of a geneticallyencoded cysteine-like amino acid is cysteine. Examples ofnon-genetically encoded cysteine-like amino acids include homocysteineand penicillamine.

As will be appreciated by those having skill in the art, the aboveclassifications are not absolute. Several amino acids exhibit more thanone characteristic property and can therefore be included in more thanone category. For example, tyrosine has both an aromatic ring and apolar hydroxyl group. Thus, tyrosine has dual properties and can beincluded in both the aromatic and polar categories. Similarly, inaddition to being able to form disulfide linkages, cysteine also has anapolar character. Thus, while not strictly classified as a hydrophobicor an apolar amino acid, in many instances, cysteine can be used toconfer hydrophobicity to a peptide.

Geobacteraceae bacteria and relatives in the order Desulfurococcalesnaturally produce protein filaments known as pili that are electricallyconductive. For this reason, they are generally referred to as microbialor pilus nanowires. The pilus nanowires are protein filaments assembledon the cell envelope through the polymerization via hydrophobicinteractions of a single peptide subunit, the pilin or PilA. Thepurified pili are electrically conductive. As the pili protrude outsidethe cell, other proteins, such as metalloproteins known asc-cytochromes, can bind the pili and may contribute to theirconductivity and adhesive properties. However, biochemical analyses ofthe purified pili have demonstrated that they were conductive withoutbeing directly associated with metals or metalloenzymes. Furthermore,they lack any biological redox cofactors such as flavins and quinones.Thus, the conductivity of the nanowire protein filament is intrinsic tothe pilin subunits in the assembly and is not due to any redox-activecomponent that may associate with the nanowire polypeptide, such asmetals, ions, contaminants, metalloenzymes, flavins or quinones.

The peptide subunit (or pilin) in the electrically conductive pili isencoded by the pilA gene of Geobacteraceae bacteria. The product of thepilA gene can generate a peptide or PilA or pilin that can polymerizevia hydrophobic interactions to form the pilus. The Geobacteraceae pilusnanowire can electrically connect the cell with electron acceptors inits environment. This electronic connection can enable the cell to gainenergy through the transfer of metabolically-generated electrons acrosselectron transport proteins, such as c-cytochromes and othermetalloproteins of the cell envelope, and through the pilus. The piluscan serve as the main electrical connection between the cell andextracellular acceptors such as Fe(III) oxides. Geobacter sulfurreducenscan be naturally found in underground sediment where anaerobicconditions may require that an electron acceptor other than oxygen beemployed and where minerals or other electron acceptors are commonlyavailable. Thus, although Geobacter sulfurreducens can utilize oxygen asan electron acceptor, these bacteria can also transfer electrons fromtheir pili to extracellular electron acceptors such as Fe(III) oxides,resulting in insoluble Fe(III) in the environment to be reduced tosoluble Fe(II) and magnetic minerals of mixed Fe(III)-Fe(II) valencesuch as magnetite.

The pilus nanowires are dynamic filaments that can protrude and retractby polymerizing and depolymerizing the pilin subunits at the cellenvelope. Thus, several pilin peptides can be assembled to make a pilusthat can function as a nanowire. Extension and retraction events arepowered, respectively, by the PilB (pilin polymerase) and PiT (pilindepolymerase) proteins, which belong to the secretion NTPasesuperfamily. The pilus nanowires can be predominantly helical instructure. In particular, they can be composed of an α-helical corespanning the hydrophobic N-terminus region that can promote pilinpolymerization, and a short αβ-loop in the C-terminal region. Thus, thepilus nanowires from Geobacteraceae bacteria lack the long αβ-loop andextensive C-terminal globular head that other bacterial pili possess.

Pilin assembly can occur via hydrophobic interactions proceeding in ahelical fashion that may help position electroactive amino acids bymerging or bonding their atomic orbitals optimally so as to favor chargetransport along and across the nanowire.

Bacteria in the genus Geobacter can produce conductive proteinappendages of the Type IVa pilus class to discharge respiratoryelectrons onto extracellular electron acceptors such as ferric iron(Fe[III]) minerals and the uranyl cation as described in Reguera, G. etal. “Extracellular electron transfer via microbial nanowires”. Nature435, 1098-1101, (2005) and Cologgi, D. et al. “Extracellular reductionof uranium via Geobacter conductive pili as a protective cellularmechanism” Proc Natl Acad Sci USA, 108, 15248-15252 (2011), incorporatedherein by reference in their entirety.

In Type IVa pili, Geobacter pili are also assemblies of primarily onepeptide subunit (the pilin or PilA), though the structural pilins thatmake the conductive pili are shorter than canonical pilins and form anindependent line of descent with pilins from other members the orderDesulfurococcales. Assembly is however predicted to follow the conservedsteps of other bacterial Type IV pili. In one embodiment, the bacteriaGeobacter sulfurreducens synthesizes two pilin precursors (prepilins)with a long or a short signal peptide that interact for optimal pilinexport to the membrane and coregulation of cytochrome export. The twoprepilin isoforms carry the conserved recognition sequences needed forremoval of the leader peptide and N-methylation of the mature peptide bya conserved PilD prepilin peptidase. A canonical Type IV pilus apparatusspanning the multilayered cell envelope assembles the pilins verticallyon the inner membrane, exposing the base of the pilus fiber to theabundant periplasmic cytochromes to facilitate the discharge ofrespiratory electrons as described in Reguera, G. “Harnessing the powerof microbial nanowires”. Microb Biotechnol 11, 979-994, (2018),incorporated herein by reference in its entirety. Conductive piliisolated from G. sulfurreducens transport charges at rates (˜1 billionelectrons per second at biologically relevant voltages of 100 mV) twoorders of magnitude greater than the cellular rates of respirationmeasured in iron oxide cultures.

Furthermore, each cell assembles numerous pili on one side of the cell,providing many conduits for the discharge of respiratory electrons. Thisbiological strategy has been proposed to maximize access to the mostbioavailable forms of iron oxides, which are dispersed in soils andsediments and rapidly transition into more crystalline and lessbioavailable mineral forms abiotically. The reduction of iron oxidessolubilizes part of the Fe(III) but also generates magnetite, a magneticmineral of mixed Fe(III)/Fe(II) that remains bound to the pilus fibers.

Similarly, the pili retain the mononuclear uranium mineral phase formedduring the reduction of the soluble uranyl cation as described inCologgi, D. L. et al., “Extracellular reduction of uranium via Geobacterconductive pili as a protective cellular mechanism” Proc Natl Acad SciUSA 108, 15248-15252, (2011), incorporated herein by reference in itsentirety. To enable new rounds of respiration, cells detach the reducedminerals by depolymerizing the pilins in a reaction energized by aconserved PilT ATPase (PilT4). The retraction of the pili stores thepilin peptides in the inner membrane, making them readily available fora new round of polymerization energized by a conserved PilB ATPase asdescribed in Speers, A. M., et al. Genetic identification of a PilTmotor in Geobacter sulfurreducens reveals a role for pilus retraction inextracellular electron transfer. Frontiers in Microbiology 7, 1578,(2016) and Steidl, R. et al. “Mechanistic stratification inelectroactive biofilms of Geobacter sulfurreducens mediated by pilusnanowires”. Nat. Commun. 7, 12217, (2016).

Antagonistic cycles of pilus protrusion and retraction by Geobactercells can sustain respiration of extracellular electron acceptors. Thisis of special significance during the reduction of the uranyl cation,which is reductively precipitated by the pili outside the cell andprevented from traversing the outer membrane.

Studies in G. sulfurreducens have also helped define structural featuresof the Geobacter pilins that are critical for fiber formation andconductivity. The reduced size of Geobacter pilins (61 amino acids in G.sulfurreducens compared to 142-175 in other bacterial pilins) is theresult of a carboxy-terminal (C-t) truncation in the conserved modulararchitecture of other Type IVa pilins. The globular head of Type IVapilins, with its distinctive αβ-loop, anti-parallel β-sheet domain andD-region flanked by two conserved cysteines, is replaced in Geobacterpilins by a short, flexible random-coiled segment. This changes thechemistry of the pilus surface and exposes on the surface of theconductive pili amino acid ligands for metal binding and reductionFeliciano, G. T. et al. “Structural and functional insights into theconductive pili of Geobacter sulfurreducens revealed in moleculardynamics simulations” Phys Chem Phys 17, 22217-22226, (2015),incorporated herein by reference in its entirety. Geobacter pilins onlyretain the amino-terminal (N-t) α-helix (α1 domain), a conformation thatpromotes electronic coupling and charge transport.

The predominantly α-helical conformation of Geobacter pilins canincrease the hydrophobicity and flexibility of the peptides compared toother pilins, allowing for tight pilin-pilin hydrophobic interactionsduring assembly and the formation of a strong yet flexible pilus fibercore. Proper alignment of pilins in the fiber core can be maintained bysalt bridges between positively and negatively charged amino acids fromneighboring al-domains. Each pilin participates in the formation of twosalt bridges (D53-K30 and D54-R28) that bend the peptide's mid-regionand align the side chains of neighboring aromatic residues(phenylalanines and tyrosines) at distances optimal for chargetransport.

FIG. 1A shows structural models of a pilus fiber and pilin peptideoptimized in Molecular Dynamics (MD) simulations showing the aromaticresidues and salt bridges that are critical for structural integrity andconductivity. MD simulations identify motions that bring some of thearomatic side chains of the pilins in the assembly within 3-5 Adistances, although these aromatic “contacts” never form at the sametime, as in a metallic wire. The geometry of the aromatic contacts isalso displaced, preventing π-π stacking and metallic-like conductivity.Yet the inter-aromatic distances and geometries of the aromatic sidechains support a coherent mechanism of conductivity. Experimentalvalidation of this prediction is available from the thermal activationof pilus conductivity demonstrated by scanning tunneling microscopy andthe low charge mobility calculated for pilus fibers purified free ofmetal and organic contaminants, which cannot support a band conductionmechanism.

In one embodiment, the present description can include a method forsynthesizing protein nanowires. The protein nanowires may be synthesizedusing peptide building blocks such as recombinant peptide buildingblocks. The recombinant peptide building blocks may be derived from theconductive pilin peptide of the electrically active bacterium Geobactersulfurreducens. The method can include nucleation and elongation stepsfor efficient peptide self-assembly and control of nanowire length. Themethod can include addition of a hydrophobe such as octadecane, providedin aqueous solution or as functionalized silica particles. The methodcan include triggering the self-assembly of the peptides and fiberformation.

The method can include increasing molecular crowding via evaporationthat can facilitate hydrophobe-guided nucleation and assembly of thepilin peptides into conductive protein fibers. The method can includeadditional refeeding steps with mixing that can be used to control thelength of the nanowires and the yields of nanowires. In one embodiment,the method can yield protein nanowires that are conductive assemblies.In one embodiment, the method can yield protein nanowires that canretain at least some or all of the biochemical and electronic propertiesof the native protein nanowires (pili) produced by Geobacter cells. Inone embodiment, the biochemical and electronic properties of the proteinnanowires are maintained under chemical fixation. This can be anadvantageous consideration for integration in electronic devices.

Microbial nanowires are described, for example, in U.S. Pat. No.8,729,233 to Reguera et al., U.S. Pat. No. 8,846,890 to Reguera et al.,and U.S. Pat. No. 9,409,955 to Reguera et al., which patents areincorporated herein by reference. Harnessing the unique properties ofGeobacter pili will ultimately require protocols for their productionand functionalization at high yields and costs at that are needed tosatisfy market demands. Direct purification of conductive pili fromnative cells is achievable yet requires many purification steps toseparate the pili from other cellular components. Moreover, cultivationof piliated cells under anaerobic conditions may not be easily scalableand yields of pure pili are low (in the mg range). Chemical synthesis ofthe naturally occurring peptides is possible, but random aggregation ofthe highly hydrophobic peptides reduces production yields and samplequality. Addition of solubility tags for recombinant expression of thenaturally occurring pilin peptides also faces challenges because thehigh hydrophobicity of the peptide causes aggregation and toxic effectsin heterologous hosts such as Escherichia coli.

In one embodiment, modified PilA peptides such as truncated versions ofthe PilA peptides at the N-terminus can overcome these challenges andenable their recombinant production when fused to suitable solubilitymodules. In one embodiment, thiolated versions of pilins engineered witha 19-amino acid N-t truncation can be successfully synthesized viarecombinant techniques and can attach to and spontaneously assembly as amonolayer onto gold electrodes. The planar assembly of the thiolatedpilins can be conductive via pilus-like mechanisms that alternate chargehopping through aromatic contacts and interchain tunneling acrossaromatic-free regions. The assembly can also expose the metal-bindingligands to the solvent and allows the monolayers to bind and reducecationic metals, like the pilus fiber. In one embodiment, the thiolatedpilins can retain the structural features and critical amino acidsneeded for self-assembly, conductivity and metal binding and reduction,making them attractive building blocks for the manufacturing of novelconductive biomaterials.

In one embodiment, a method of synthesizing protein nanowires isprovided. The protein nanowires can be conductive nanowires. In oneembodiment, the protein nanowires can be synthesized from peptidebuilding blocks. In one embodiment, the peptide building blocks caninclude pilin peptides. The pilin peptides can be assembled to formpilus or pili nanowires that can be conductive. The nanowires formedfrom the peptide building blocks can be assembled to be conductive bytransfer of electrons to a substrate such as iron cation, uranyl cationand the like.

In one embodiment, the method can include providing purified peptidebuilding blocks. In one embodiment, the peptide building blocks can berecombinant peptides that have been expressed in a recombinant hostsystem. The recombinant host system can be, for example, an E.coli-based host system. Other host systems may also be used and arewithin the scope of this description. In some embodiments, therecombinant peptide building blocks are overexpressed and can result inhigh amounts of peptide building blocks that can be isolated and/orpurified. In one embodiment, the peptide building blocks can besynthesized, modified PilA peptides.

Bottom up fabrication of protein nanowires using recombinant pilinpeptides using evaporation of a peptide containing solution wasdescribed in U.S. Pat. No. 9,601,227 to Reguera, G., et al. andincorporated herein by reference in its entirety.

The methods described herein include a hydrophobe, refeeding stepsand/or reaction mixing to control the yields and length of the nanowireproduct. The methods described herein have been optimized to control thecritical assembly, increase production yields of nanowires with customlengths, and reduce sample-sample variability.

In one embodiment, the present description can include a method forscalable production and purification of recombinant pilins whose shortN-t truncations can reduce their hydrophobicity without perturbing thestructural and biochemical motifs critical for self-assembly andconductivity. The methods described herein also can include protocolsfor the bottom-up self-assembly of pilins into protein nanowires havingstructural and electronic characteristics similar to those of naturallyoccurring pili purified from G. sulfurreducens.

Methods for synthesis of nanowires will be described with reference torecombinant PilA peptides but it will be understood that synthesizedPilA peptides may also be used in the methods for the nanowire synthesisand are within the scope of this description.

Unlike the synthesis of inorganic semiconductors, the bottom-upfabrication of pilin-based nanowires does not require complex crystalgrowth or the use of toxic metals. The methods described herein relyinstead on a hydrophobe-triggered nucleation step and an elongation stepthat controls the length of the nanowire product. This self-assemblyprotocol, and the genetic amenability of the recombinant productionsystem, offer opportunities to tune the properties of the peptide, andconsequently, the functional characteristics of the resulting nanowireto design novel protein-based conductive nanomaterials forbioelectronics and other applications.

As shown in FIG. 1B, pilins can be designed with short truncations atthe N-terminus of PilA pilin peptide to reduce the hydrophobicity of thepeptides while preserving structural features and amino acids criticalfor self-assembly into conductive protein fibers. Computational analysesof the amino acid sequence of the native PilA pilin peptide (SEQ IDNO:1) of G. sulfurreducens via AGGRESCAN identified two regions in thepeptide (residues 1-22 and 25-31) as having highest propensity toaggregate, with the first 10 amino acids contributing the most (FIG.1B). In one embodiment, a truncation of about 11 amino acids can benecessary to reverse the sign of the GRAVY score of the PilA frompositive to negative, a proxy for solubility. In one embodiment, theKyte Doolittle plot can localize the highest hydrophobicity within thefirst 21 amino acids of PilA.

In some embodiments, truncations can be from about 10 amino acids toabout 21 amino acids of native PilA peptide may lead to solubilizationof the truncated PilA peptides and can facilitate the recombinantproduction of the synthesized nanowires. In some embodiments, thesetruncations can also be within the ranges that can preserve the aromaticand charged residues of the pilin required for fiber formation andconductivity (FIG. 1B). In one embodiment, the N-terminus region can betargeted to engineer pilin derivatives that can be suitable forrecombinant expression using a previously described E. coli recombinantpilin production system Cosert, K. M., et al. “Electroniccharacterization of Geobacter sulfurreducens pilins in self-assembledmonolayers unmasks tunneling and hopping conduction pathways”. Phys ChemChem Phys 19, 11163-11172, (2017), incorporated herein by reference inits entirety.

In one embodiment, the recombinant peptides or peptide building blockscan be expressed as a fusion polypeptide.

Nucleic acids encoding peptide building blocks such as truncated PilApeptides, can be used for recombinant expression of the nanowirepeptides, for example, by operably-linking the peptide building blocknucleic acid to an expression control sequence within an expressionvector, which can be introduced into a host cell for expression of theencoded peptide building blocks. The nucleic acids that encode peptidebuilding blocks can also encode a fusion partner fused in-frame with thepeptide building blocks, for example, to facilitate expression orpurification of the peptide building blocks.

A nucleic acid molecule encoding a peptide building blocks canoptionally be optimized for expression in a particular host cell andthen operably linked to one or more transcription regulatory sequences,e.g., a promoter, one or more enhancers, a transcription terminationsequence or a combination thereof, to form an expression cassette.

The peptide building blocks can also be expressed as fusion proteins. Toexpress the peptide building blocks as a fusion protein, the nucleicacids that encode the peptide building blocks can also encode a fusionpartner fused in-frame with the peptide building blocks. The fusionpartner can serve as solubility and affinity tag to the peptide tofacilitate its expression and purification. For example, fusionexpression systems may add to the peptides a His tag (allowingpurification on a Nickel column; Clontech Laboratories, Inc., Qiagen,Life Technologies Corp.); a MalE maltose binding protein, (New EnglandBiolabs, allowing purification on an amylose column); a thioredoxin(allowing purification with a phenyl arsine oxide resin); aglutathione-S-transferase (GST, allowing purification with glutathione)and a chitin binding domain (allowing purification with chitin columns,New England Biolabs). By also encoding a signal peptide in-frame withthe fusion protein some of these systems (e.g., MalE, His Tag™ (Roche))can be adapted for periplasmic expression. Cytoplasmic expression can beachieved with these systems when no signal peptide is incorporated. Theexpressed fusion protein can contain a specific protease cleavage sitefor cleavage and removal of the fusion partner peptide. It may alsoinclude a self-splicing element such as an intein linker, which releasesthe peptide in the presence of a reducing agent such as DTT.

The type of fusion partner peptide can influence the ease or extent ofexpression and purification. For example, some types of fusion partnerpeptides may interfere with, or promote folding, aggregation,degradation, or solubility of the fusion protein. In general, a fusionpartner peptide is selected that facilitates fusion protein expression,folding, solubility, purification or any combination thereof. In someembodiments, the fusion partner peptide can protect the fusion proteinfrom proteolytic digestion or inhibit proteolytic degradation.

One example of a fusion partner peptide that is useful for expressionand production of peptide building blocks is the chitin binding domain(CBD). The small size (about 5-7 kDa), substrate binding specificity andhigh avidity of CBDs for chitin has led to their utilization as affinitytags for immobilization of proteins to chitin surfaces (Bernard, M. P.,et al. Anal. Biochem. 327:278-283 (2004); Ferrandon, S., et al. Biochim.Biophys. Acta. 1621: 31-40 (2003)). For example, the B. circulanschitinase A1 type 3 CBD has been used to immobilize fusion proteinsexpressed in bacteria on chitin beads to provide a platform forintein-mediated protein splicing (Ferrandon, S., et al., Biochim.Biophys. Acta. 1621: 31-40 (2003)) and to chitin-coated microtiterdishes (Bernard, M. P., et al., Anal. Biochem. 327:278-283 (2004)).

CBD as a component of chitinase can be obtained from many differentsources, for example, fungi, bacteria, plants and insects. Any CBDoriginating from a chitinase may be used herein although CBDs separatedfrom chitinase catalytic activity are preferred.

Nucleic acids encoding peptide building blocks (or fusion proteins) canbe incorporated into bacterial, viral, insect, yeast or mammalianexpression vectors so that they are operably linked to expressioncontrol sequences such as bacterial, viral, insect, yeast or mammalianpromoters (or enhancers).

Nucleic acid molecules or expression cassette that encode peptidebuilding blocks (or fusion proteins) may be introduced to a vector,e.g., a plasmid or viral vector, which optionally includes a selectablemarker gene, and the vector introduced to a cell of interest, forexample, a bacterial, yeast or mammalian host cell.

In one embodiment, the fusion polypeptides can be, for example, thepeptide building blocks fused to an affinity tag polypeptide and alinker chitin binding domain (CBD) polypeptide. In these embodiments,the fusion polypeptide is expressed as a soluble complex. The affinitytag can be used to purify the fusion polypeptides by, for example,passing the culture lysates expressing the fusion polypeptide through anaffinity chromatography column. The fusion polypeptides can bind to thecolumn and can then later be eluted. Splicing of the fusion polypeptideat the linker can result in a composition that can include purifiedpeptide building block. Methods of isolating truncated polypeptidesusing a recombinant expression system with fusion polypeptides aredescribed, for example, in Cosert, K. M., et al. Phys Chem Chem Phys 19,11163-11172, (2017), incorporated herein by reference in its entirety.Other expression system using other affinity tags may also be used andare within the scope of this description.

In one embodiment, the recombinant platform in the recombinantexpression system can tag the pilin's N-terminus with a self-splicingintein linker and/or a solubility and affinity tag, e.g. Chitin BindingDomain or CBD. This system can facilitate the expression of therecombinant truncated PilA peptides in the soluble fraction collectedfrom culture lysates and purification by affinity chromatography.

Examples of host cells useful for manufacture of peptide building blockscan include, but are not limited to, E. coli, Salmonella species,Bacillus species, Streptomyces species, and the like, plant cells, e.g.Arabidopsis species, Taxus species, Catharanthus species, Nicotianaspecies, Oryza species, soybeans, alfalfa, tomatoes, and the like),fungal cells (e.g., Kluyveromyces species, Saccharomyces species, Pichiaspecies, Hansenula species, Yarrowia species, Neurospora species,Aspergillus species, Penicillium species, Candida species,Schizosaccharomyces species, Cryptococcus species, Coprinus species,Ustilago species, Magnaporth species, Trichoderma species, and thelike), insect cells (e.g., Sf9 cells, Sf12 cells, Trichoplusia in cells,Drosophila species and the like), or mammalian cells (e.g., primary celllines, HeLa cells, NSO cells, BHK cells, HEK-293 cells, PER-C6 cells,and the like). These cells may be grown in cultures ranging frommicroliter volumes to multiliter volumes.

In some embodiments, the peptide building blocks can include truncatedPilA peptides. Truncated PilA peptides can be PilA peptides in which oneor more amino acids are deleted. In one embodiment, the deletion ofamino acids can be at the N-terminus end of the PilA peptides. Withoutbeing bound by any theory, it is understood that deletion of some or allof the hydrophobic region of the PilA peptide can increase thesolubility of the peptide. In one embodiment, deletion of a portion ofthe N-terminal end can increase the solubility of the truncated PilApeptide while maintaining the ability of the truncated PilA polypeptideto form pilin nanowires that retain conductivity. In one embodiment, theability to form nanowires and retain conductivity can be maintained bypreserving the aromatic and charged residues found in the full-lengthnative PilA peptide (SEQ ID NO:1).

In truncated PilA peptides, amino acids can be removed from theN-terminus or the C-terminus. In general, the N-terminus of nanowirepeptides is more hydrophobic than the C-terminus, and the amino acidsthat participate in intramolecular and intermolecular electron transferprocesses across and along the pilus nanowires are located closer to theC-terminus.

In some embodiments, one or more amino acids may be removed from theN-terminus of the pilin subunit. In other embodiments, two or more,three or more, four or more, five or more, six or more, seven or more,eight or more, nine or more, ten or more, eleven or more, twelve ormore, thirteen or more, fourteen or more, fifteen or more, sixteen ormore, seventeen or more, eighteen or more, nineteen or more, twenty ormore, twenty one or more, or twenty two or more amino acids are removedfrom a nanowire peptide.

In some embodiments, the truncations may include stepwise codonreductions of the amino-terminus of the PilA peptides to reduce thesubunit hydrophobicity and improve its expression in a heterologoushost. The truncations generally do not affect amino acids shown to beinvolved in electron transfer and metal binding and are optimized topreserve the subunit ability to assemble via hydrophobic interactions.

In some embodiments, a string of amino acids can also be removed fromnanowire peptides. For example, a sequential segment of about 1 to about4 amino acids can be removed, or a sequential segment of about 1 toabout 5 amino acids can be removed, or a sequential segment of about 1to about 7 amino acids can be removed, or a sequential segment of about1 to about 10 amino acids can be removed, or a sequential segment ofabout 1 to about 12 amino acids can be removed, or a sequential segmentof about 1 to about 14 amino acids can be removed, or a sequentialsegment of about 1 to about 15 amino acids can be removed, or asequential segment of about 1 to about 16 amino acids can be removed, ora sequential segment of about 1 to about 17 amino acids can be removed,or a sequential segment of about 1 to about 18 amino acids can beremoved, or a sequential segment of about 1 to about 19 amino acids canbe removed, or a sequential segment of about 1 to about 20 amino acidscan be removed, or a sequential segment of about 1 to about 21 aminoacids can be removed, or a sequential segment of about 1 to about 22amino acids can be removed, or a sequential segment of about 1 to about23 amino acids can be removed, or a sequential segment of about 1 toabout 24 amino acids can be removed, or a sequential segment of about 1to about 25 amino acids can be removed.

In one embodiment, the truncated PilA peptides can have a deletionwithin the first 22 amino acids of the PilA peptide. In someembodiments, truncated PilA peptides may have a truncation of about 10to about 22 amino acids for solubility. In one embodiment, truncatedPilA peptide can have a deletion of the first 10 amino acids (PilA₁₀).In one embodiment, truncated PilA peptide can have a deletion of thefirst 19 amino acids (PilA₁₉). In one embodiment, truncated PilA peptidecan have a deletion of the first 20 amino acids (PilA₂₀). In oneembodiment, truncated PilA peptide can have a deletion of the first 22amino acids (PilA₂₂).

In some embodiments, the truncation may be within the hydrophobic regionbut may retain some of the terminal amino acids. For example, thetruncated PilA peptides may have a truncation of from about amino acid 3to about amino acid 20 of the PilA peptide, or from about amino acid 5to about amino acid 20 of the PilA peptide, or from about amino acid 10to about amino acid 22, further including any range therebetween. Othertruncations are also possible and are within the scope of thisdescription.

In one embodiment, this approach can recover PilA peptide buildingblocks in the soluble fraction of culture lysates fusion proteinscontaining pilins engineered with truncations of 10, 19, 20 and 22 aminoacids as shown below.

PilA (SEQ ID NO: 1) N-term- FTLIELLIVV AIIGILAAIA IPQFSAYRVKAYNSAASSDL RNLKTALESA FADDQTYPPE S PilA₁₀ (SEQ ID NO: 2)N-term- AIIGILAAIA IPQFSAYRVK AYNSAASSDL RNLKTALESA FADDQTYPPE S PilA₁₉(SEQ ID NO: 3) N-term AIPQFSAYRV KAYNSAASSD LRNLKTALES AFADDQTYPP ESPilA₂₀ (SEQ ID NO: 4) N-term- IPQFSAYRVK AYNSAASSDLRNLKTALESA FADDQTYPPE S PilA₂₂ (SEQ ID NO: 5)N-term- QFSAYRVKAY NSAASSDLRN LKTALESAFA DDQTYPPES

FIG. 1B shows the alignment of pilA peptide (SEQ ID NO:1) aligned withPilA₁₀ (SEQ ID NO:2), PilA₁₉ (SEQ ID NO:3) PilA₂₀ (SEQ ID NO:4) andPilA₂₂ (SEQ ID NO:5).

In some embodiments, the conductive pili can have truncated pilinpeptides with at least about 10%, or at least about 20%, or at leastabout 30%, or at least about 45%, or at least about 50%, or at leastabout 55%, or at least about 60%, or at least about 65%, or at leastabout 70%, or at least about 75%, or at least about 80%, or at leastabout 85%, or at least about 90%, or at least about 95%, or at leastabout 97% sequence identity to a pilin peptide having an amino acidsequence comprising any of the SEQ ID NO:1-5.

In further embodiments, the truncated pilin peptides with at least 60%sequence identity of the SEQ ID NO:1 can have a truncation at theN-terminus of about 30 amino acids, or of about 1-28 amino acids, or ofabout 1-25 amino acids, or of about 1-22 amino acids, or of about 1-20amino acids, or of about 1-19 amino acids, or of about 1-17 amino acids,or of about 1-15 amino acids, or of about 1-13 amino acids, or of about1-12 amino acids, or of about 1-10 amino acids, or of about 1-9 aminoacids, or of about 1-8 amino acids, or of about 1-7 amino acids, or ofabout 1-6 amino acids, or of about 1-5 amino acids, or of about 1-4amino acids, or of about 1-3 amino acids, or of about 1-2 amino acids.

Chemical synthesis (to synthesize peptides de novo), chemicalmodification (e.g., which may include chemical stripping) or geneticengineering, can be used to manipulate the peptide composition,structure and binding properties of microbial nanowires to selectivelymodify conductance properties. Microbial nanowires can also bemanipulated via genetic engineering to bind specific ligands for sensordesign, controlled and specific deposition during device manufacturing,and the like. In one embodiment, genetic engineering or chemicalmodification is used to produce nanowires with various functionalities.In one embodiment, Geobacter sulfurreducens, is used. For additionaldetails and sequence listings, see U.S. application Ser. No. 13/221,495,filed on Aug. 30, 2011 and entitled, “Microbial Nanowires,” both ofwhich are hereby incorporated by reference herein in their entireties.

In some embodiments, the peptide building blocks may be chemicallysynthesized and/or modified. Chemically modified peptide building blocksand polypeptides can be generated from nanowire peptides/polypeptideswith a natural (non-recombinantly engineered) sequence that ischemically modified. In other embodiments, the chemically modifiedpeptide building blocks can be generated with a mutant nanowire peptidethat contains substitutions, deletions or additions of amino acids thatare not normally found in naturally occurring pilus nanowires. Thus, forexample, before chemical modification, the peptide building blocks canhave a variant or modification thereof.

In some embodiments, the peptide building blocks can be chemicallysynthesized or modified after their synthesis to modulate theconductive, adhesive, coupling or other properties of the synthesizednanowires. The chemical modification may be performed on the peptidebuilding block or after the synthesis on nanowires. Such chemicalmodification can be performed by procedures available in the art using avariety of reagents. For example, reagents such as performic acid,peroxides, iodoacetamide, iodoacetic acid, bissulfosuccinimidyl suberate(BS3), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, N-ethylmaleimide,methyl methanethiosulfonate andS-(2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl)methylmethanesulfonothioate (MTSL) can be used to modify the conductive,adhesive, coupling or other properties of the nanowire polypeptides. Inother embodiments, the nanowire peptides or polypeptides can beglycosylated, acylated or conjugated to an alkylene glycol (e.g.,polyethylene glycol or PEG). Such modifications can be performed byprocedures available in the art. See, e.g., John M. Walker, The ProteinProtocols Handbook (2002); Means, G. E. and Feeney, R. E. ChemicalModifications of Proteins. Holden-Day, San Francisco (1971).

In one embodiment, the PilA₁₉ can be used as a peptide building block ina method for the synthesis of protein nanowires. In one embodiment,peptide building blocks that can be solubilized in a buffer may be usedas peptide building blocks in the synthesis of protein nanowires. Thedescription herein discloses the synthesis of protein nanowires withPilA₁₉ as the peptide building block. It will be understood that otherpeptide building blocks such as other truncated PilA peptides may alsobe used, and all are within the scope of this description.

In one embodiment, the truncated PilA peptides can be separated fromother proteins in a chitin column as shown in FIG. 2. Truncated Pil Apeptides with 10, 19, 20 and 22 amino acids removed from the N-terminuswill be referred to herein as PilA₁₀, PilA₁₉, PilA₂₀ and PilA₂₂,respectively.

In one embodiment, truncating 19 amino acids (PilA₁₉) can reverse thesign of the grand average hydropathy (GRAVY) score of the PilA₁₉ peptidefrom (+0.51) to (−0.55). This can be indicative of the solubility of thetruncated peptides and can permit the high-yield recovery of thetruncated PilA peptides, e.g. PilA₁₉ peptide, after cleavage from theCBD tag as shown in FIG. 2. In one embodiment, the cleavage efficiencycan be highest for PilA₁₉ than for other soluble peptides such as PilA₂₀and PilA₂₂. This can allow for the nearly complete recovery of thepeptide after cleavage for 24 h at room temperature as shown in FIG. 2.This can be due to the fusion of the 19 amino acid truncation peptide tothe intein linker via a residue (alanine as shown in FIG. 1) that can beoptimal for DTT-induced cleavage.

The peptide building blocks can be purified by any available method. Inone embodiment, the method includes lysis of cells expressing thepeptide building blocks, followed by selective removal of contaminatingcell macromolecules, and then selective separation of pure peptidebuilding blocks from other proteins. In one embodiment, a single steppurification method is used which may have yields in excess of 50%, suchas up to 55% or up to 60% or higher, including any and all ranges therebetween. In one embodiment, the yield is at least about 63%. Higheryields, in excess of 63% may also be possible, such as up to about 95%,including any and all ranges there between. The protocol is flexible, inthe sense that it can be adapted for use with substantially any sampleof peptide building block expressing cells, substantially any method toremove contaminating cell macromolecules that do not affect theintegrity of the peptide building blocks, and substantially any methodto selectively separate the peptide building blocks from othercontaminating proteins.

In some embodiments, the host cells are used to mass-produce the peptidebuilding blocks and then purified for self-assembly of the nanowires orpili as described herein. In some embodiments, the fusion partnerpeptide is a chitin binding domain. When the fusion partner peptide is achitin binding domain, a matrix or solid substrate containing acarbohydrate can be used, where the carbohydrate is bound by the chitinbinding domain. For example, the chitin binding domain(s) can bindchitin. Chitin can be linked, adsorbed or covalently bound to a solidsubstrate such as a bead, column matrix or a coated surface. The solidsubstrate may, for example, magnetic chitin beads, colloidal chitin orenvironmental chitin. The chitin may also be immobilized in a column orcoated on a solid surface. In one example, sterile chitin beads areadded directly to culture medium so that protein production andharvesting can occur simultaneously during the fermentation process(see, e.g., U.S. Pat. No. 7,732,565 or U.S. Patent ApplicationPublication. No. 2006/041849, both of which are hereby incorporated byreference herein in their entireties).

Once the solid substrate (e.g., beads, matrix or a column) has beenwashed to remove contaminating molecules, the peptide building blockscan be obtained by cleaving a bond linking the nanowire peptide to thefusion partner peptide, then washing the peptide from the solidsubstrate, leaving the fusion partner peptide attached to the solidsubstrate. It may be desirable to elute the peptide building blocks fromthe matrix under non-denaturing conditions.

For example, a fusion protein that includes a fusion partner peptidethat binds to a binding entity can be affinity purified by contactingthe fusion protein with a solid substrate to which the binding entity isabsorbed or bound. After binding to the fusion protein to a solidsubstrate, the fusion partner peptide can be cleaved, and the peptidebuilding blocks can be washed from the solid substrate. The fusionpartner peptide can be retained by the solid substrate. For example, thepeptide building blocks can be cleaved from a CBD fusion partner peptideby washing the solid substrate with a reducing agent such asβ-mercapoethanol or dithiothreitol.

In some embodiments, the expression of the recombinant fusion proteins,e.g. pilA peptide-CBD, can be similar with all the truncated pilins. Insome embodiments, the amount of peptide that can be eluted from thecolumn after DTT cleavage of the CBD can vary widely as shown in FIG. 2.In one embodiment, the cleavage efficiency via intein self-splicing canbe sensitive to the peptide residues adjacent to the intein linker andthe temperature during the elution from the column. In one embodiment,increasing the temperature from about 4° C. to about 23° C., forexample, can improve the cleavage efficiency for all the PilA_(n)peptides. In one embodiment, increasing the temperature can also promotethe aggregation of the hydrophobic peptide, e.g. PilA₁₀ after cleavage.As a result, yields of PilA₁₀ in solution can be too low to detect thepeptide band in an SDS-PAGE gel. In one embodiment, low yields ofPilA_(n), such as PilA₁₀, can be detected by MALDI-TOF mass spectrometryas shown in FIG. 3.

In one embodiment shown in FIG. 4A, a recombinant production cycle ofPilA₁₉ building blocks is shown with the enrichment of the CBD-PilA₁₉fusion protein in soluble fractions collected from replicate culturelysates and the purification of the PilA₁₉ peptide after incubating thechitin-bound CBD PilA₁₉ protein with DTT at room temperature (23° C.)for 24 h.

In one embodiment, the peptide building blocks can be evaluated withrespect to the secondary structure and folding dynamics. A variety ofmethods can be used to evaluate the secondary structure of the peptidebuilding blocks and all are within the scope of this description.

In one embodiment, circular dichroism (CD) can be used to investigatethe secondary structure and folding dynamics of PilA₁₉. (FIG. 4B). Inone embodiment, maintaining a helical conformation can be advantageousfor pilin peptides to establish hydrophobic interactions during fiberformation. In one embodiment, a detergent such as sodium dodecyl sulfate(SDS) can be used to determine the formation of α-helical conformationsof a truncated pilin polypeptide and suitability for fiber formation. Inone embodiment, the ellipticity of a truncated polypeptide may bemeasured to determine the formation of an ordered structure such as anα-helix.

In one embodiment, the methods described herein can include in vitroself-assembly of the peptide building blocks to produce filamentsanalogous to the native ones that have customizable structural andfunctional properties. In some embodiments, genetic engineering can alsobe used to add functional groups to the peptide building blocks, (e.g.,for enhancing manufacture, folding, assembly, binding and other usefulproperties (e.g., including allowing synthesis of functionalizednanowires in nanostructured interfaces).

In one embodiment, only one type of purified peptide building block canbe provided during self-assembly for formation of recombinant nanowires.In one embodiment, a mixture of two or more purified peptide buildingblocks can be provided during self-assembly for formation of recombinantnanowires. In one embodiment, different peptide building blocks can beprovided simultaneously or separately at the initial assembly reactionor during refeeding of the assembly components as described below duringsynthesis of nanowires.

In one embodiment, the method further includes suspending the purifiedpeptide building blocks, e.g. truncated pilin peptides, in an assemblybuffer. Assembly buffer can be any solvent or combination of solventsthat can maintain the peptide building blocks in solution and/or thepeptide structure and/or charge that is optimal for nanowire formation.In one embodiment, the assembly buffer can include a solvent that isless than polar than water. In one embodiment, the assembly buffer caninclude a solvent that stabilizes the peptide's helical structure.Assembly buffer can include, for example, acetonitrile, methanol or acombination of the two. Other solvents may also be used and are withinthe scope of this description.

In one embodiment, the assembly buffer can include a combination ofsolvents. In one embodiment, the assembly buffer can include an organicsolvent such as acetonitrile that can maintain the peptide buildingblocks in solution. In one embodiment, the assembly buffer can includeother solvents, such as methanol, that can stabilize helical peptideconformations. Other solvents that stabilize helical peptideconformations can include, for example, trifluoroethanol.

In one embodiment, the assembly buffer can include acetonitrile andmethanol. In one embodiment, the assembly buffer can be anacetonitrile:methanol having a ratio of about 90:10, or about 80:20, orabout 70:30, or about 60:40, or about 50:50, or about 40:60, or about30:70, or about 20:80, or about 10:90. Other combinations of solventsmay be used that can maintain the peptide building blocks in solution.In one embodiment, the assembly buffer can be acetonitrile:methanolhaving a ratio of about 80:20.

In some embodiments, assembly buffer may comprises an aqueous solutionof methanol or trifluoroethanol. In one embodiment, assembly bufferswith chemically synthesized peptides may include solvents with water.

The concentration of the peptide building blocks in the assembly buffercan vary. In one embodiment, the concentration of the peptide buildingblocks in the assembly buffer is at least about 3 mg/ml. In oneembodiment, the concentration of the peptide building blocks in theassembly buffer is from about 3 mg/ml to about 15 mg/ml. In oneembodiment, the concentration of the peptide building blocks in theassembly buffer is from about 7 mg/ml to about 10 mg/ml.

In one embodiment, the method can include initiating nucleation ofpeptide building blocks to self-assemble and promote the self-assemblyof the peptide building blocks in the assembly buffer to form fibers.The method can include providing a hydrophobe in the assembly buffer toform an assembly composition. In one embodiment, the assemblycomposition can include the peptide building blocks and a hydrophobe inthe assembly buffer. Hydrophobes can be molecules and/or materials thatcan serve as the nucleating site and can guide self-assembly of peptidebuilding blocks into protein fibers.

In one embodiment, the hydrophobe can include long chain carbonmolecules. In one embodiment, the hydrophobe can include long chaincarbon non-polar molecules. In one embodiment, the hydrophobe caninclude at least about 8 carbons, or at least about 10 carbons, or atleast about 13 carbons, or at least about 15 carbons, or at least about17 carbons, or at least about 18 carbons, or at least about 20 carbonsin the carbon molecules.

In one embodiment, the hydrophobe can be a hydrophobic chemicalmolecule. In one embodiment, the hydrophobe can be an alkane. In oneembodiment, the hydrophobe can be chemical molecules, such as adetergent, dextran, methacrylate and the like, added directly to thesolvent or in surface constrained forms such as coatings on silica oragarose beads. In one embodiment, the hydrophobe is octadecane.

In one embodiment, the hydrophobe can be added to the assembly bufferwith the peptide building blocks. The amount of the hydrophobe that canbe included can vary and may be dependent on the amount and type of thepeptide building blocks. In one embodiment, the ratio of hydrophobe topeptide building block is at least about 1:20. In one embodiment, theratio of hydrophobe to peptide building block is from about 1:20 toabout 1:500, or from about 1:50 to about 1:200, or from about 1:80 toabout 1:120. In one embodiment, the ratio of hydrophobe to peptidebuilding block is about 1:100.

In one embodiment, an optimal concentration of the peptide may beestablished first. Then the concentration of the peptide can be keptconstant to calculate the concentrations of hydrophobe that can promotefiber formation.

In one embodiment, the method of providing a hydrophobe can include theuse of a column to perform a buffer exchange by transferring the peptidebuilding blocks from an elution buffer to an assembly buffer. In oneembodiment, elution buffer can elute the peptide from the chitin columnafter DTT-induced splicing from the CBD-intein module.

In one embodiment, the buffer exchange can lead to the co-elution of thepeptide building blocks with a hydrophobe into the assembly buffer. Inone embodiment, the buffer exchange column can include particles coatedwith long chain carbon molecules.

In one embodiment, the hydrophobe can be a material such as particlescoated with long chain carbon molecules. These particles can be theparticles in the buffer exchange column. The long chain carbon moleculescan be non-polar carbon molecules. In one embodiment, the hydrophobe caninclude, for example, silica particles. Other particles can function ashydrophobes such as for example, agarose, which is used extensively tomake hydrophobic resins such as Superdex© (dextran cross-linked withagarose beads). In some embodiment, particles made with hydrophobicepoxy resins may also be included as a hydrophobe. In one embodiment,the silica particles can be coated with long chain carbon molecules. Inone embodiment, the hydrophobe can be octadecyl-silica particles. Othermaterials that can be used to make beads of the desired size and coatedwith a hydrophobic ligand such as an alkane. Beads can be, for example,from about 0.25 um to about 2 um. In one embodiment, the beads can befrom about 0.5 um to about 1 um.

In one embodiment, the buffer exchange column can be a reverse phasecolumn. In one embodiment, the reverse phase column can include silicaparticles. In one embodiment, the reverse phase column can includeoctadecyl-silica particles.

In one embodiment, the method can include loading a buffer exchangecolumn with peptide building blocks. In one embodiment, the peptidebuilding blocks can be in an elution buffer. In one embodiment, thepeptide building blocks can be soluble in the elution buffer aftercleavage of the affinity tag from the recombinant host system. In otherwords, the elution buffer can be a buffer that can maintain the purifiedpeptide building blocks in solution.

In one embodiment, after loading the peptide building blocks for thebuffer exchange, the column can be washed with water or aresin-compatible buffer. The washing can remove the impurities. Theassembly buffer can be added to detach the peptide from the resin forcollection in a tube. In one embodiment, the column may be washed withabout 5 ml of liquid, or from about 5 ml to about 10 ml of liquid, orfrom about 10 ml to about 15 ml of liquid, or from about 15 ml to about20 ml of liquid, or from about 20 ml to about 25 ml of liquid, or fromabout 25 ml to about 30 ml of liquid. The liquid for washing the columncan be a buffer, water and the like.

In one embodiment, a buffer exchange may be conducted to transfer thepeptide building blocks from the elution buffer (from the chitin column)to the assembly buffer. In one embodiment, after the peptide buildingblocks are loaded onto the buffer exchange column, the peptide buildingblocks can be eluted into the assembly buffer.

In one embodiment, the peptide building blocks can be loaded onto thecolumn in elution buffer (from the chitin column). The peptide buildingblocks are retained in the column and the elution buffer may be washedaway using other buffers or water. The buffer exchange column with thebound peptide blocks can be washed with water. Assembly buffer can thenbe added to the column to detach the peptide building blocks. Thus, thepeptide building blocks go into the column in elution buffer and comeout in assembly buffer resulting in a buffer exchange.

In one embodiment, the elution of the peptide building blocks can alsolead to co-elution of the buffer exchange column particles into theassembly buffer. In one embodiment, the coated particles from the bufferexchange column can serve as the hydrophobe in the assembly composition.In one embodiment, the loading of the column, washing and elution can beperformed using a gravity flow rate.

In one embodiment, the pH of the peptide building block composition atthe time of loading is about 9 and after elution is about neutral. ThepH of the composition at the time of loading and elution can vary. OtherpH's at the time of loading and elution are also within the scope ofthis description.

The concentration of the peptide building blocks in the assembly bufferafter elution can vary. In one embodiment, the concentration of thepeptide building blocks in the assembly buffer after elution can be fromabout 1 mg peptide/ml to about 8 mg/ml. In one embodiment, theconcentration of the peptide building blocks in the assembly bufferafter elution can be from about 2 mg peptide/ml to about 5 mgpeptide/ml. In one embodiment, the concentration of the peptide buildingblocks in the assembly buffer after elution can be about 3 mgpeptide/ml.

The amount of the coated column particles in the assembly compositioncan vary. In one embodiment, the amount of coated column particles canbe from about 1 mM to about 10 mM in the assembly composition. In oneembodiment, the amount of coated column particles can be from about 2 mMto about 5 mM in the assembly composition. In one embodiment, the amountof coated column particles can be from about 3 mM to about 4 mM in theassembly composition. In one embodiment, the amount of coated columnparticles can be about 3.5 mM in the assembly composition.

In one embodiment, the method can further include evaporating theassembly buffer in the assembly composition after the addition of ahyrophobe. In one embodiment, the assembly composition may be formed bythe addition of a hydrophobe to the assembly buffer containing thepeptide building blocks. In one embodiment, the assembly composition maybe formed by co-elution of the hydrophobe with the peptide buildingblocks. Evaporation of the assembly buffer from the assembly compositioncan increase the molecular crowding in the assembly composition. Withoutbeing bound by any theory, it is believed that increase in molecularcrowding can create a hydrophobic environment that can promotepeptide-peptide interactions and lead to assembly of the pilin peptidesinto fibers.

A variety of methods can be used to promote evaporation of the assemblybuffer. In one embodiment, the evaporation of the assembly buffer can beperformed by a SpeedVac Concentrator. In one embodiment, evaporation ofthe assembly buffer can occur by centrifugation of the tube with theassembly composition. This can be conducted with the tube open, e.g. notsealed, so that evaporation of the assembly buffer can occur. Anexemplary method of evaporation is described below in the Examples. Itwill be understood other methods are known in the art and may be used topromote evaporation of the liquid from the assembly composition.

In one embodiment, a portion of the assembly buffer can evaporate fromthe assembly composition. In one embodiment, all of the assembly buffercan evaporate from the assembly composition. In one embodiment, at leastabout 25% of the assembly buffer may evaporate, or at least about 50% ofthe assembly buffer may evaporate, or at least about 75% of the assemblybuffer may evaporate, or at least about 85% of the assembly buffer mayevaporate, or at least about 90% of the assembly buffer may evaporate,or at least about 95% of the assembly buffer may evaporate.

In one embodiment, the method can include performing one or moreelongation cycles. In one embodiment, an elongation cycle can includerefeeding the assembly composition with additional peptide buildingblocks after evaporation, mixing the assembly composition afterrefeeding and evaporating the liquid in the assembly composition.

In one embodiment, the method can include two or more elongation cycles.In one embodiment, the method can include three or more elongationcycles. In one embodiment, the method can include from about 3elongation cycles to about 6 elongation cycles. In one embodiment, themethod can include from about 3 elongation cycles to about 5 elongationcycles. In one embodiment, the method can include about 4 elongationcycles.

In one embodiment, the elongation cycle can include refeeding theassembly composition with additional peptide building blocks. In oneembodiment, refeeding may include addition of hydrophobe and/oradditional assembly buffer to the assembly composition. Refeeding canprovide additional building blocks for generating fibers and/ornanowires. It can also increase molecular crowding to further promotefibers and/or nanowires.

In one embodiment, the same peptide building blocks can be added duringeach refeeding step. In one embodiment, different peptide buildingblocks may be added during the refeeding step. In one embodiment,peptide building blocks can include a mixture of different peptidebuilding blocks. In one embodiment the peptide building block mixturecan include 2, or 3, or 4, or 5 or more, or 10 or more, or 15 or moredifferent peptides in the peptide building block mix. In one embodiment,the same peptide building block mixture can be added during eachrefeeding step. In one embodiment, different peptide building blockmixture can be added during each refeeding step.

In one embodiment, the method can further include mixing the compositionafter the refeeding with peptide building blocks and assembly buffer.Mixing can resuspend the peptides and/or fibers already formed and canincrease the interaction between the newly added peptide building blocksand the peptides and/or fibers already present in the assemblycomposition. This can allow the interaction and incorporation of thenewly added peptide building blocks with peptides and/or fibers alreadypresent in the assembly composition to further elongate and/or widen thepeptides and/or fibers.

In one embodiment, mixing can be done by aspirating the assemblycomposition in and out of a micropipette multiple times to disperse thecomponents in the assembly composition. In one embodiment, the tube orvessel with the assembly composition can be vortexed to disperse thecomponents in the assembly composition. In one exemplary embodiment,mixing may be performed as described in the Examples below or any typeof mechanical agitation. Other methods for mixing the components areknown in the art may also be used and all are within the scope of thisdescription.

In one embodiment, the method can include harvesting the synthesizednanowires by processing after the last elongation cycle. The processingsteps can include, for example, drying the synthesized nanowirecomposition, resuspending the nanowire composition, precipitating thenanowire composition, and/or lyopholization may be performed. In oneembodiment, precipitation of the fibers by acetone may be performed toremove the silica beads. In one embodiment, nanowires formed in theassembly composition can be dried after the last elongation cycle hasoccurred. The dried assembly composition can be resuspended in a liquidsuch as water or buffer. In one embodiment, the nanowires may beprecipitated by acetone, o/n and recovered by centrifugation. In oneembodiment, the nanowires can be dried under a stream of N2 and storedfor further use.

In one embodiment, FIG. 11 shows a schematic diagram of thepolymerization of peptide building blocks to form recombinant nanowires.The pilins polymerized in vitro can follow kinetics that fit the typicalnucleation-dependent polymerization model. Nucleation-controlledaggregation kinetics can include an initial lag phase of molecularorganization and peptide nucleation that can be accelerated in thepresence of seed molecules such as hydrophobes. A linear phase of fibergrowth then follows until reaching a saturation or stationary phase,which marks the equilibrium between soluble monomers and fibers and theend of fiber growth. As shown in FIG. 11, the lag phase can be minimizedby suspending the peptide building block monomers in an organic solventthat stabilized the peptide's helical conformation and adding ahydrophobe that provided seed molecules to trigger nucleation and guidefiber growth. The controlled evaporation of the solvent can increasemolecular crowding and promote the initial nucleation of the pilins onthe seed or hydrophobe molecules (primary nucleation) and theirspontaneous self-assembly as short fibers (secondary nucleation). Fiberelongation depended on the availability of hydrophobe and peptidebuilding blocks supplied in subsequent refeeding steps but also onreaction mixing, which increased the number of nucleation sites and theincorporation of monomers into the growing fibers until reaching thesaturation phase.

Octadecane, whether in solution (FIG. 9A-9B) or immobilized on silicaparticles (FIG. 5A), can be a suitable hydrophobe to trigger pilinnucleation and fiber formation. This can suggest that the hydrophobedoes not need to be incorporated into the fiber but, rather, it providesa physical site to nucleate the pilins and guide their self-assembly asfibers (FIG. 11). The addition of the hydrophobe in asurface-constrained form can also permit its separation from the fibersby centrifugation at the end of the assembly reaction. The emission ofsilica from nanosized particles in a precise region of the UV spectrum(FIG. 6A) also proved useful to optimize the concentration ofhydrophobe.

In one embodiment, at optimal hydrophobe concentrations, and withsufficient peptide refeeding and reaction mixing steps, PilA₁₉ fiberswere synthesized approximately 6-μm long that dispersed well in mildaqueous solutions (FIG. 5A-5C). This contrasts with purificationprotocols available for native pili, whose longer and heterogeneouslength promotes the formation of large supramolecular structures thatare difficult to disrupt without denaturing the pilus fiber core (FIG.5A). The reduced aggregation of the PilA₁₉ fibers can also facilitatetheir deposition on electrode surfaces and electronic characterizationby scanning probe methods (FIG. 10A-10H).

In one embodiment, the nanowires synthesized according to the methodsdescribed herein can be produced in large quantities using a simpleprotocol relative to the natural nanowires.

In one embodiment, nanowires having a length of at least about 1 μm canbe generated. In one embodiment, nanowires having a length from about 4μm to about 10 μm can be generated. In one embodiment, nanowires havinga length from about 5 μm to about 7 μm can be generated.

In one embodiment, synthesized nanowires having an average diameter ofat least 1 nm can be generated. In one embodiment, synthesized nanowireshaving an average diameter of from about 1 nm to about 3 nm can begenerated. In one embodiment, synthesized nanowires having an averagediameter of about 2 nm can be generated.

In one embodiment, the synthesized fibers and nanowires can becharacterized by a variety of methods and techniques.

In one embodiment, the fibers can be characterized by performingcircular dichroism (CD) spectra on a sample of the fibers and/ornanowires. The ratio of the intensity at 222 nm and 208 nm can be usedto indicate the helical content of the sample. In one embodiment, theratio of 222 nm/208 nm is from about 0.7 to about 0.9. In oneembodiment, the ratio of 222 nm/208 nm is from about 0.9 to about 2 orhigher.

In one embodiment, the nanowires can be characterized by performingscanning probe microscopy. In one embodiment, the fibers or nanowirescan be deposited on the surface of Highly Oriented Pyrolytic Graphite(HOPG) as described in the Examples below. In one embodiment, thenanowires can have rectifying behavior. In one embodiment, analyses ofthe asymmetry of the IV plots can have a rectification score below about1.

In one embodiment, the nanowires can be chemically fixed prior to use indevices as described herein.

In one embodiment, the synthesized nanowires can transfer electrons tometal cations. The nanowires can transfer electrons to a variety of ionsincluding, for example, Fe(III), uranyl cation and the like.

The nanowire proteins synthesized by assembly of modified PilA peptidescan be essentially pure. Synthesized nanowires can be free ofcontaminants, metals, ions, metalloenzymes, flavins, quinones and otherredox cofactors that can be found in purified, naturally occurringnanowires. In one embodiment, the synthesized nanowires can be composedof truncated PilA peptides that polymerize according to the methodsdescribed herein via hydrophobic interactions to form the pilus, i.e.,synthesized nanowire filament. These nanowires can be stored drysubstantially indefinitely and can be resuspended in appropriatesolvents, as needed, for downstream applications. As noted herein, thesenovel synthesized nanowires have conductive (e.g., rectifying) behaviordue, in part, to the polarized nature of proteins, containing anN-terminus (positively charged) and a C-terminus (negatively charged)end. Particular rectifying behavior can also be due to the proteincomposition (i.e., amino acid make-up) and structure of the nanowire(i.e., due to the alignment of dipoles of peptide bonds in the pilin'sα-helix).

The nanowire polypeptides described herein can have asymmetricalconducting properties due to the protein composition (i.e., amino acidmake-up) and structure of the nanowire. Such conducting activity canalso be characteristic of the disclosed nanowire peptides andpolypeptides in pure form, for example, in absence of metals andcellular contaminants that could mask the natural rectifying propertiesof the nanowire.

By combining more than one asymmetric conductor together, a device canbe made with a variety of conductive properties. Moreover, theconductive properties of such a device can be altered by employinggenetically or chemically modified nanowire peptides and or byincorporating other materials available to those of skill in the art.

Devices that include microbial nanowires are desirable because thenanowire peptides can be mass-produced and induced to self-assemble toproduce the nanowires. This can enable the mass-production ofnanowire-containing devices at a low cost.

The disclosed synthesized nanowires may be used in various deviceapplications such as antenna, attenuator, battery, brush, capacitor,condenser, conductor, circuit, electrode, fuel cell, generator, filtercircuit breaker (fuse), inductor, coil, nanowire array, particlecollector, precipitator, reactor, rectifier, relay, resistor, solarenergy collector, spark generator, suppressor, terminal, and the like.

The synthesized nanowires can also be used, for example, forconstruction of active devices such as transistors. With regard tonano-electronics, the conductive behavior of nanowires means thatprotein-based diodes (one-way conductors) can be constructed from thesenanowires. In conventional microelectronics, diodes are the basicbuilding blocks for transistors and more complex active components,including the microprocessors that run our computers. Hence, theconducting behavior of the nanowires opens the door to the constructionof protein-based nano-electronics transistors and more complex devices.

In one embodiment, nano-electronics include, for example, radiodemodulation (rectification of AM radio frequency signals to make audiosignals), low voltage AC-DC power conversion, current steering, powerswitches and over-voltage protection. Other embodiments include, but arenot limited to, the logic circuitry in electronic devices such as laptopcomputers, cellular phones and similar devices, further includingcomputer chips, such as those used in the transportation industry, suchas in aircraft and automobiles.

In some embodiments, the nanowire polypeptides can be configured toinclude branches, for example, by chemically linking two or morenanowire peptides or polypeptides together. Thus, the nanowirepolypeptides can be assembled into a main pilus that is elongated andhas a selected or desirable length. A plurality of branch pili mayemanate from the main nanowire pilus at one or more substantially fixeddistances along the length of the main pilus. The main pilus may alsoinclude one or more junctions with one or more secondary main pili,where the junctions are substantially perpendicular to the length of themain pilus. In some embodiments, junctions may be developed by growingor expressing nanowire peptides between two regions that may beelectrically connected.

In another embodiment, the nanowire peptides can be configured to formpart of an apparatus. For example, the apparatus may contain at leastone pilus having truncated nanowires peptides. In other embodiments, theapparatus may contain at least one junction between pili. For example,the apparatus may include a plurality of junctions. Each junction mayinclude a branch pilus and an elongate main pilus. For example, eachjunction may be situated at an interface between a branch pilus and theelongate main pilus.

In one embodiment, the disclosed pili are used in the design andfabrication of biobased devices. Such devices may include biosensors,biocatalytic systems, biofuel cells, and heavy-metal transformationsystems and the like. In some embodiments the pilin monomers or pili maybe interfaced with a suitable substrate such as gold or carbon.Substrates suitable for fabricating bioelectronic interfaces includesubstrates that allow nanowires to self-assemble and be conductive.

In some embodiments, the synthesized nanowires can be used to bind andreductively precipitate toxic contaminants such as uranium along thenanowires.

Embodiments of the invention will be further described by reference tothe following examples, which are offered to further illustrate variousembodiments of the present invention. It should be understood, however,that many variations and modifications may be made while remainingwithin the scope of the present invention.

EXAMPLES

Materials and Methods

Bacterial strains and culture conditions. Geobacter sulfurreducensstrain PCA was routinely grown in anaerobic NB medium with 20 mM acetateas electron donor and 40 mM fumarate as electron acceptor. Genomic DNAextracted from these cultures was used as template to PCR-amplify thenative pilA gene (GSU1496) and engineer recombinant pilin productionsystems in E. coli Rosetta™ 2 (DE3) pLysS cells (Novagen), as describedbelow. The E. coli cultures were propagated in Luria Bertani (LB) mediumsupplemented with antibiotics, as described below, and preserved in 20%glycerol at −80° C.

Design, recombinant production and purification of truncated pilins. Thetruncated pilins used in this study (PilA₁₀, (SEQ ID NO:2); PilA₁₉, (SEQID NO:3); PilA₂₀, (SEQ ID NO:4); and PilA₂₂, (SEQ ID NO:5)) arederivatives of the mature PilA peptide of G. sulfurreducens carrying 10,19, 20 and 22 amino acid truncations at the peptide's N-t. Thetruncation design was based on analyses of the hydrophobic regions andaggregation potential of the PilA peptide (i.e., the pilin without itssignal peptide) based on computational predictions with AGGRESCAN, grandaverage hydropathy (GRAVY) scoring, and Kyte Doolittle test. Alltruncations preserved the aromatic and charged amino acids of the pilinthat are critical for conductivity and formation of salt bridges. Pilindesign also involved computational analyses of MD-optimized structuralmodels of the PilA pilus fiber (GPIL-WT.pdb) and PilA pilin(pilin-WT.pdb) constructed with the MacPyMOL: PyMOL v1.8.2.2 softwareenhanced for Mac OS X (SchroÅNdinger LLC). Pilin truncations wereintroduced with PCR primers (Table 1) using as a template the pilA gene(GSU1496) of G. sulfurreducens cloned in the pTYB11 plasmid vector(IMPACT™-CN system, New England Biolabs). Table 1 shows primers (forwardand reverse) used to clone the mature pilA sequence in the expressionvector pTYB11 and PCR-amplified truncated derivatives (pilA_(n)). Theresulting plasmids (pTYB11::pilAn, where n stand for the number of N-tamino acids truncated) were transformed into E. coli Rosetta™ 2 (DE3)pLysS cells (Novagen) for recombinant expression of the PilA_(n) peptidefused at the N-t to an intein linker and a chitin-binding domain (CBD),as described elsewhere.

TABLE 1 Primer sequence Plasmid (5′-3′)¹ SEQ ID NO. pTYB11::pilAGGTGGTTGCTCTTCCAACT SEQ ID NO. 6 TCACCCTTATCGAGCTGCT GGTGGTCTGCAGTCATTAASEQ ID NO. 7 CTTTCGGGCGGATAGGT pTYB11::pilA10 GGTGGTCTGCAGTCATTAASEQ ID NO. 8 CTTTCGGGCGGATAGGT GGTGGTTGCTCTTCCAACG SEQ ID NO. 9CGATCATCGGTATTCTCGC pTYB11::pilA19 GGTGGTTGCTCTTCCAACG SEQ ID NO. 10CGATTCCGCAGTTCTCGGC GGTGGTCTGCAGTCATTAA SEQ ID NO. 11 CTTTCGGGCGGATAGGTpTYB11::pilA20 GGTGGTTGCTCTTCCAACA SEQ ID NO. 12 TTCCGCAGTTCTCGGCGTAGGTGGTCTGCAGTCATTAA SEQ ID NO. 13 CTTTCGGGCGGATAGGT pTYB11::pilA22 GGTGGTTGCTCTTCCAACC SEQ ID NO. 14 AGTTCTCGGCGTATCGTGTGGTGGTCTGCAGTCATTAA SEQ ID NO. 15 CTTTCGGGCGGATAGGT ¹Restriction sites(SapI, GCTCTTC; PstI, CTGCAG) are underlined.

Recombinant expression of CBD-PilA_(n) fusion proteins in the E. colihost was in 1 L cultures of LB broth supplemented with 100 μg/mlampicillin and 20 μg/ml chloramphenicol and incubated at 37° C. to anOD₆₀₀˜0.4 before induction with 50 mM of isopropylβ-D-1-thiogalactopyranoside (IPTG) during overnight incubation at 16°C., as previously described. Cells harvested by centrifugation (4,000×gfor 10 min) were resuspended in 20 mM Tris-HCl buffer (100 mM NaCl, 1 mMEDTA, 1% CHAPS) and lysed by tip sonication. Centrifugation of thelysate (12,000×g for 30 min at 4° C.) separated the soluble proteinswith the fusion protein in the supernatant fraction. Purification of thefusion protein from the other soluble proteins was by affinitychromatography in a chitin column (New England Biolabs; ca. 40 ml bedvolume) equilibrated with 200 ml of column buffer (20 mM Tris, 100 mMNaCl, 1 mM EDTA, pH 7.4). After incubation with the soluble proteinfraction at room temperature for 20 min to promote the attachment of thefusion protein to the chitin matrix, we washed the column with 200 ml ofbuffer at increasing salt concentrations (20 mM Tris, 1 mM EDTA, 0.6/1 MNaCl pH 7.4) to remove the unbound proteins.

Cleavage of the PilA_(n) peptides from the chitin-bound fusion proteinwas by induction of intein self-splicing with 50 mM 1,4-dithiothreitol(DTT). To do this, the column was incubated with ˜200 ml of cleavagebuffer (20 mM Tris, 100 mM NaCl, 50 mM DTT, pH 9) for 24, 48 or 72 h atroom temperature (23° C.) or at 4° C. to minimize the aggregation of thepeptide once cleaved from the solubility tag. A column wash with thesame buffer but without DTT (elution buffer) eluted the PilA peptides in2-ml eluent fractions, which we identified by measuring the absorbanceat 280 nm of the eluted fractions. Because some of the peptidesaggregated after elution, cleavage efficiency was estimated from theratio of the CBD over the full CBD-PilA_(n) proteins retained in thechitin column after DTT cleavage and elution of the peptide. To do this,the chitin beads were removed from the column and resolubilized thechitin-bound proteins in 1% SDS at 100° C. Separation of the solubilizedproteins was in a 7.5% Tris/glycine SDS-PAGE (Bio-rad) run for 30 min at200 V in Tris/glycine/SDS buffer (25 mM Tris, 192 mM glycine, 0.1% w/vSDS) using a Bio-rad Mini Trans-Blot cell system. The proteins in thegels were stained with Bio-safe Coomassie (Bio-rad, Hercules, Calif.)for 1 h and destained in ddH₂O. The density of the fast migrating band(CBD module) and the slower migrating CBD-PilAn protein band was used tocalculate the percent of CBD-PilA_(n) that was cleaved.

Cleavage at room temperature for 24 h was optimal for high-yieldcleavage of PilA₁₉ and used thereafter. Peptide-containing fractionswere pooled before estimating the protein concentration by absorbance at280 nm using a NanoDrop™ spectrophotometer (Thermo Scientific). SDS-PAGEwas used to monitor the recombinant expression of the fusion protein(CBD-PilA_(n)) and purity of the PilA peptide eluting from the chitincolumn using 10-20% Tris-Tricine polyacrylamide gels run for 75-120 minat 100V in Tris/tricine/SDS buffer (100 mM Tris, 100 mM Tricine, 0.1%w/v SDS). Proteins in the tricine gels were fixed for 30 min with anaqueous solution of 50% methanol and 40% acetic acid prior to stainingwith Bio-safe Coomassie (Bio-rad, Hercules, Calif.) for 1 h andde-staining with ddH₂O until bands were visible. The mass of the peptidewas confirmed by Matrix Assisted Laser Desorption-Ionization-Time ofFlight (MALDI-TOF) mass spectrometry using a 1 μl peptide solution mixedwith 1 μl of 50 mM 3,5-dimethoxy-4-hydroxycinnamic acid in 50%acetonitrile CH₃CN/0.5% trifluroacetic acid (TFA) and dried on a sampleplate. Mass spectra collection was on a time-of-flight (TOF) Voyager-DEPro-MALDI-TOF mass spectrometer (Applied Biosystems, Framingham, Mass.).

Circular Dichroism (CD). PilA₁₉ peptides purified in elution buffer weredialyzed against 10 mM potassium acetate buffer (with 50 mM Na₂SO₄, pH3.8) using Spectra/Por® Biotech cellulose ester dialysis membranes (MWCO100-500 Da). The peptide concentration was determined from thedifference spectrum (320 to 270 nm) of the protein dissolved in 1 ml of6 M guanidine hydrochloride at pH 12.5 versus pH 7.1 (39) and with theknown molar extinction coefficients of tyrosine and tryptophan residues(40) using the equation:

$\begin{matrix}{c = \frac{A_{293}}{{2\text{,}357\; Y} + {830\; W}}} & \left( {{eq}.\mspace{14mu} 1} \right)\end{matrix}$

where A₂₉₃ is the absorbance at 293 nm in the difference spectrum, Y isthe number of tyrosine (3 in PilA₁₉) and W is the number of tryptophan(0 in PilA₁₉) residues.

The concentration of the peptide in the buffer was adjusted toapproximately 50 μg/ml prior to CD spectroscopy. When indicated, sodiumdodecyl sulfate (SDS) was added to the peptide solution at a finalconcentration of 1, 8 or 40 mM. The peptide solutions were dispensed ina quartz cuvette (0.1 cm path length, Starna Cells Inc.) and their CDspectrum in the 190 to 360 nm was collected at 0.5 nm increments (5second integration time) using a Chirascan™ spectrometer (AppliedPhotophysics Ltd., Leatherhead, United Kingdom). The spectra werebaseline-corrected and smoothed using a third order Savitsky-Golayfilter. The CD instrument units (θ, millidegrees) were converted intomean residue molar ellipticity [θ] units using the Wallace and Janesequation (41):

$\begin{matrix}{\lbrack\theta\rbrack = \left( \frac{\theta \times {0.1} \times {MRW}}{c \times l} \right)} & \left( {{eq}.\mspace{14mu} 2} \right)\end{matrix}$

where c is the peptide concentration in mg/ml, l is the path length ofthe cuvette in cm (0.1 cm), and MRW is the mean residue weight of thesample estimated from the molecular mass MW in Daltons (4,524 Da forPilA₁₉) and the number n of amino acid residues (42 for PilA₁₉), asfollows:

$\begin{matrix}{{MRW} = \frac{M\; W}{n - 1}} & \left( {{eq}.\mspace{14mu} 3} \right)\end{matrix}$

The CD data was used to estimate the α-helix content of peptide usingthe program CONTINLL at the DICROWEB server. The program is amodification of the CONTIN method that uses a ridge regression algorithmto estimate the CD spectrum of unknown proteins by comparison to alinear combination of CD spectra of N reference proteins with knownconformations. Because the reference proteins are predominantlyglobular, the conformation estimates for peptides, fibrous proteins, andmembrane proteins are approximate. The program evaluates the goodness offit parameter normalized mean residue standard deviation (NMRSD), whichis defined as:

$\begin{matrix}{{NRMSD}{= \left\lbrack \frac{\sum\left( {\theta_{\exp} - \theta_{cal}} \right)^{2}}{\sum\left( \theta_{\exp} \right)^{2}} \right\rbrack^{1/2}}} & (3.4)\end{matrix}$

where θ_(exp) and θ_(cal) are the experimental and calculatedellipticity values at a specific wavelength. A NRMSD value of less than0.1 is generally considered a good fit. Thus, NRMSD values above 0.1were rejected.

The CD spectra were also collected for fibers assembled with recombinantPilA₁₉ peptides resuspended for 30 min in 10 mM potassium acetate with50 mM Na₂SO₄ (pH 7). A 500 μl aliquot of a 40 μg/ml (Nanodrop estimate)of the pili solution was dispensed into a quartz cuvette with a 1 mmpath length (Starna Cells Inc., Atascadero, Calif.) and scanned from 190to 260 nm at 0.5 nm increments with a 5 second integration time withautomated baseline subtraction. Native pili purified from G.sulfurreducens were used as controls before or after denaturation with 8M urea. All the scans were adjusted from θ, millidegrees, to molarellipticity using equation, as described above.

In vitro assembly of PilA₁₉ pilins. PilA₁₉ fibers were synthesized in aprotocol that incorporated a buffer exchange step to resuspend thepeptides in assembly buffer (80:20, acetonitrile:methanol) andevaporation-induced assembly in the presence of a hydrophobe. Thestandard protocol for maximum yields of fiber formation with PilA₁₉ useda reverse phase C18 column (Sep-Pak C18 3 cc Vac Cartridge, 55-105 μmParticle Size, Waters Corporation, Milford, Mass.) for buffer exchangeinside an anaerobic chamber (COY Labs). The resin in the cartridges wasfirst hydrated with 5 ml of acetonitrile and equilibrated with 5 ml ofddH₂O, following manufacturer's recommendations. A solution of therecombinant peptide (8 mg of PilA₁₉ in 10 ml of elution buffer) wasapplied to the column by gravity flow and the peptide retained in theC18 resin was washed with 5 ml of ddH₂O (1.7× column volume) beforeelution in a disposable glass tube with 3 ml of a freshly preparedassembly buffer. When indicated, the washing step was extended from 5 to9, 12, 15 or 18 or 15 ml of ddH₂O. DTT and C18-silica particlesco-eluting with the peptide were identified in the UV-vis spectrum ofthe solution as absorbance peaks at 205 nm (DTT) and 245 nm (silica)using a Shimadzu UV-2401PC spectrophotometer. Control solutions withpure silica particles demonstrated the sensitivity of the detectionmethod to particle sizes of less than 1 m (FIGS. 6C-6D). Standard ofpure silica particles 0.5-1 m in diameter were used to calculate theconcentration of C18-silica particles in the solution.

When indicated the buffer exchange step was carried out in an Oasis Max™extraction cartridge (60 mg of non-silanol polymeric sorbentfunctionalized with a quaternary amine sorbent, Waters Corporation,Milford, Mass.). The column was hydrated with 1 ml of acetonitrile andwashed with 1 ml of ddH₂O before loading the 2 ml of the peptidesolution previously adjusted to a pH of 10 with 1 mM NaOH. After washingthe column with 1 ml of 5% NH₄OH, the peptide was eluted with 1 mlassembly buffer and the UV-vis spectrum was collected to analyze thecolumn eluant, as described for the C18-column eluants. Peptides elutedfrom these columns required the addition of a hydrophobe (octadecane,1:100 aqueous solution) to promote fiber formation.

The standard protocol for evaporation-induced self-assembly started witha 1-ml aliquot of the peptide solution (˜8 mg) containing the hydrophobe(e.g., 1:100 octadecane or C18-silica particles) in assembly buffer anda first round of evaporation for 30 min at 45° C. in a Savant™ SpeedVac™concentrator (SPD121P model, Thermo Fisher). Fiber elongation wascontrolled through refeeding steps every 30 minutes (4 times with 500 μlof the peptide solution) and reaction mixing by aspiration with amicropipette. At the end of the evaporation process, the dried samplewas resuspended in 200 μl of ddH₂O and dispensed in 50 μl aliquots.Addition of 200 μl ice cold acetone to each aliquot and overnightincubation at −20° C. precipitated the fibers, allowing to recover themby centrifugation (1 h, 4° C. in a microcentrifuge). After a finaldrying step under a stream of N₂, the sample was stored at −20° C. untilfurther use. When indicated, the final drying step was vialyophilization in order to measure the average particle size of thesample via dynamic light scattering (DLS) in a Malvern Zetasizer Nano-ZS(0.3 nm to 10 microns sensitivity). Assembly efficiency was alsocalculated as the difference of free PilA₁₉ monomer in solution beforeand after assembly, based on protein concentrations measured byabsorbance at 280 nm in a NanoDrop™ spectrophotometer (ThermoScientific) using a standard curve of bovine serum albumin (BSA).

Scanning Probe Microscopy. Conductivity studies were conducted in aclean room using PilA₁₉ fibers stored dried at −20° C. and rehydrated in200 μl of ddH₂O at 4° C. for 12-18 h. Deposition for 10 minutes of 10aliquots of the solution onto the surface of a freshly-cleaved HighlyOriented Pyrolytic Graphite (HOPG; SPI Supplies) promoted the adsorptionof the fibers to the electrode. Absorbent lens paper wicked off excessfluid while two washes with 10 of ddH₂O removed impurities from the HOPGsurface. Samples were allowed to dry in a sealed container at roomtemperature for approximately 10 minutes before imaging the samples intapping mode by Atomic Force Microscopy (AFM) with an Asylum ResearchCypher S system equipped with an AC240TS tip (Asylum Research).Electrode surface scans were typically of 10×10 μm² to locate the areaof sample deposition and image several fields randomly that bestrepresented the distribution of fibers on the surface. The AFM imageswere analysed with the free hand tool of ImageJ to measure the length ofthe fibers. Conductive probe AFM (CP-AFM) analyses, which measures thetransversal conductivity of the sample from the electrons flowingbetween the conductive AFM tip and the HOPG electrode, followedprotocols previously used to measure the conductivity of the nativepili. As controls, we also conducted AFM topographic and CP-AFMconductivity analyses of the native PilA pili, which were purified asreported elsewhere. The ±100 mV ohmic region in the IV plots was fittedto a linear regression line using the Igor Pro 6 software to calculatethe electrical resistance of the fibers at biological voltages. Analysesof the asymmetry of the IV plots was via rectification scores (ratio ofcurrent recorded at the positive over the negative voltage) calculatedat voltages comparable or exceeding the biological-relevant voltage (100and 600 mV, respectively) as reported elsewhere. A rectification scorebelow 1 indicates asymmetric current flow that favors theelectrode-to-tip direction, thus electrons flowing from the fibertowards the external electron acceptor.

Scanning Tunneling Microscopy (STM) analyses (imaging and spectroscopy)used the same AFM instrument but equipped with a mechanically cut Pt:IrSTM tip (Asylum Research) and operated in STM mode. The quality of theSTM tip was tested in scans on the freshly cleaved HOPG surface prior todepositing and scanning the pili samples (sample voltage of 500 mV;current set point of 350 pA). Sample deposition was with hydrated orglutaraldehyde-fixed PilA₁₉ fibers, using protocols described for thedeposition of hydrated or chemically-fixed native PilA pili. IV plotscollected the current tunneling through individual fibers at a set pointof 10 pA, with the tip held at ground, while sweeping the bias voltage(±0.6 V) of the HOPG substrate. The linearity of the plot in the ±100 mVohmic region was analyzed by the fit of a linear regression line withthe Igor Pro 6 software. The asymmetry of the STM IV plots and thematerial's electron band gap were assessed in plots of the derivative ofthe current and voltage data points (dI/dV) versus the sample voltage(V).

Example 1—Design, Recombinant Production and Structural Characterizationof Pilin Building Blocks

Computational analyses of the PilA sequence via AGGRESCAN (14)identified two regions in the peptide (residues 1-22 and 25-31) withhighest aggregation propensity (FIG. 1B). Aggregation scores wereparticularly high for the first 10 amino acids, which are also among themost hydrophobic residues identified in a Kyte Doolittle plot (15) (FIG.1B). The aggregation and hydrophobicity analyses predict truncations ofthe first 21-22 amino acids as having the highest impact on solubility.As this extended truncation preserves the aromatic and charged residuesrequired for fiber formation and conductivity (FIG. 1B), this N-t regionwas targeted to engineer pilin derivatives suitable for recombinantexpression with a self-splicing intein linker and a solubility andaffinity tag (Chitin Binding Domain or CBD). The recombinant approachreproducibly recovered in the soluble fraction of culture lysates fusionproteins containing pilins engineered with truncations of 10, 19, 20 and22 amino acids (FIG. 1B-D).

Affinity chromatography in a chitin column retained the fusion proteinsbound to the chitin matrix and permitted the elution of the pilinpeptide after inducing the self-splicing of the intein linker withdithiothreitol (DTT) (FIG. 4A). FIG. 4A shows, as an example, theenrichment of the CBD-PilA₁₉ fusion protein in soluble fractionscollected from replicate culture lysates and the purification of thePilA₁₉ peptide after incubating the chitin-bound CBD-PilA₁₉ protein withDTT at room temperature (23° C.) for 24 h.

While the amount of recombinant fusion proteins recovered from thesoluble culture fractions was similar for all the truncated pilins, theamount of peptide eluted from the affinity column after DTT cleavagevaried widely (FIG. 2). Cleavage efficiency via intein self-splicing issensitive to temperature and to the peptide residues adjacent to theintein linker. Increasing the temperature from 4° C. to 23° C., forexample, improved the cleavage efficiency for all the PilA_(n) peptidesbut also promoted the aggregation of the most hydrophobic peptide(PilA₁₀) once cleaved. As a result, the amount of PilA₁₀ recovered insolution was too low for visualization by SDS-PAGE and requireddetection via MALDI-TOF mass spectrometry (FIG. 3).

By contrast, truncating 19 amino acids (PilA₁₉) permitted the high-yieldrecovery of the PilA₁₉ peptide after cleavage from the CBD tag (FIG.4A). The 19-amino acid truncation fused the peptide to the intein linkervia a residue (alanine) (FIG. 1B) that is optimal for inteinself-splicing. As a result, cleavage efficiency was higher for PilA₉than for any other soluble peptides (PilA₂₀ and PilA₂₂), allowing forthe nearly complete recovery of the peptide in the column eluant (FIG.2). In addition to enabling the highest biosynthetic yields, PilA₁₉retained the helical conformation that is critical for self-assembly andfiber formation once in the presence of a hydrophobe. The foldingdynamics of the PilA₁₉ was investigated by circular dichroism (CD) as afunction of the concentration of a detergent such as SDS in the peptidesolution (FIG. 4B). The far UV CD spectrum of the peptide in 10 mMpotassium acetate buffer at pH 7 showed very low ellipticity above 210nm and a strong negative signal around 200 nm, consistent with adisordered peptide. But addition of SDS shifted the CD spectrum andrevealed the characteristic maxima (at ˜190 nm) and minima (at ˜208 and˜222 nm) of α-helical conformations. Furthermore, the intensity of thepositive (190 nm) and negative (208 nm and 222 nm) helical signalsreached maxima at or above the critical micellar concentration (CMC) ofthe detergent (˜8 mM in water) (19, 20) (FIG. 4C). At this thresholdconcentration, SDS recreates the hydrophobic environment of the innermembrane, where the pilins are stored prior to assembly to stabilizetheir α-helical conformation.

Concentrations at or above the CMC (8 mM and 40 mM) also producedintensity ratios of 222 nm over 208 nm (˜0.7) close to the 0.8 ratioexpected for a single-stranded α-helix (22). As the peptide has anegative net charge of −1.1 at neutral pH, we minimized electrostaticeffects by collecting CD spectra of control solutions at a pH of 3.8. Atthis acidic pH, below the theoretical isoelectric point (pI, 4.86) ofPilA₁₉, the peptide has a net positive charge of +3.2 that cancels outelectrostatic repulsion forces with the anionic detergent. The intensityof the α-helical signature peaks was greater at pH 3.8 than at pH 7(FIG. 4C). The more favorable electrostatic interactions between thepeptide and the detergent at the acidic pH also increased the pilin'shelical content (from ˜45% to 56% at or above the CMC and from 27% to˜49% below the CMC). The increased helical content of the PilA₁₉ peptideat the acidic pH also increased the intensity ratio of the 222 nm and208 nm peaks to 1, consistent with the assembly of 2 or more α-helicesin coiled coil configurations. These results demonstrated that PilA₁₉can adopt the helical conformation that is critical for pilinself-assembly and electronic coupling in the pilus fiber. Additionally,the studies highlighted the critical role that hydrophobicity andelectrostatics have in modulating the folding and self-assembly of thepeptides.

Example 2—Fiber Formation Via Self-Assembly of PilA₁₉ Peptides

The helical folding of PilA₁₉ and subunit assembly in solution wasinvestigated. Hydrophobic conditions optimal for controlledself-assembly of the PilA₁₉ peptides into fibers was studied. FIG. 5Ashows the main steps of the protocol optimized for bottom-up fabricationof PilA₁₉ fibers. The fabrication protocol starts with a buffer exchangestep that resuspends the peptides in a buffer of acetonitrile andmethanol suitable for evaporation-induced self-assembly in the presenceof a hydrophobe. Acetonitrile has lower polarity than water to helpmaintain recombinant pilin peptides in solution. Methanol is known tostabilize helical peptide conformations in solution. Addition of ahydrophobe triggered self-assembly of the peptides, whereas controlledevaporation of the solvent increased molecular crowding and facilitatedpeptide-peptide interactions needed for fiber formation (FIG. 5A).Octadecane, a straight-chain alkane hydrocarbon of 18 carbon atoms(C18), was a suitable hydrophobe to promote the nucleation of the pilinsand fiber formation (FIG. 9A-9B). Atomic Force Microscopy (AFM) imagingof the octadecane-triggered assembly reaction revealed, however, anextensive coating of the fibers and underlying electrode by ahydrocarbon layer that prevented conductivity measurements (FIG. 9A-9B).To bypass this limitation, the octadecane was replaced with silicaparticles functionalized with a coating of the nucleating octadecylcarbon chains (C18) (FIG. 5A-5C). By carrying out the buffer exchangestep in a reverse phase column packed with a reverse phase resin ofC18-silica particles (55-105 μm in diameter), the PilA₁₉ peptide wassimultaneously eluted and nucleating C18-silica particles approximately25-50 nm for hydrophobe-triggered fiber formation (FIG. 6A-6D).

The hydrophobe may also be added after a buffer exchange step in adifferent matrix. A buffer exchange step was conducted in an Oasis Max™exchange cartridge containing 60 mg of non-silanol polymeric sorbentfunctionalized with a quaternary amine sorbent (Waters Corporation,Milford, Mass.). The column was hydrated with 1 ml of acetonitrile andwashed with 1 ml of ddH₂O before loading the 2 ml of the peptidesolution previously adjusted to a pH of 10 with 1 mM NaOH. Thecolumn-bound peptide was washed with 1 ml solution of 5% NH₄OH andeluted with 1 ml solution of assembly buffer. FIG. 9 shows the UV-visspectrum of eluent collected from an Oasis Max™ exchange cartridge andthe induction of pilin assembly in the evaporation step with theaddition of octadecane. The UVvis spectrum shows the DTT peak carriedover with the pilins (from the affinity purification step in a chitincolumn), but no silica (˜245 nm). Addition of octadecane (1:100 aqueoussolution) to the peptide solution formed assembly composition followedby the standard evaporation/refeeding/mixing protocol (FIG. 5A) wasrequired to trigger fiber formation (FIGS. 9A-9B).

Insights into the rate-limiting steps of PilA₁₉ assembly was gained byinvestigating the effect of hydrophobe concentration in fiber formation.For these experiments, Dynamic Light Scattering (DLS) was used togrossly estimate fiber size in reactions with different concentrationsof the C18-silica particles and AFM to image the structural features ofthe assemblies (FIG. 5B). C18-silica particle concentrations of 3.5 mMtriggered fiber formation (FIG. 5B) and permitted maximum assemblyefficiencies (40-55% of the pilin monomers assembled as fibers).Efficient assembly also required reaction mixing. Thus, unmixed assemblyreactions with optimal concentrations of the nucleating C18-silicaparticles produced fibers 1(±0.5)-μm long and assembly efficiencies aslow as 14% (FIG. 5C). However, mixing the assembly reactions byaspiration with a micropipette during the peptide refeeding stepspromoted the assembly of the pilins and increased fiber length to6(±1)-μm long (FIG. 5C). This suggests that the initial evaporation stepof a 1 ml volume of assembly buffer with the peptide (˜3 mg) stimulatedpilin nucleation by the C18-silica hydrophobe, and four sequentialpeptide refeeding steps (total of 6 mg of peptide) with mixing increasedthe number of nucleation sites available for peptide assembly and theavailability of peptide building blocks to grow the fibers. Controlswith mixing but suboptimal concentrations of the hydrophobe (0.2 mM)resulted in the formation of small aggregates interspersed with shortfibers (FIG. 5B). Thus, fiber elongation is both dependent on hydrophobeconcentration and reaction mixing. Additional refeeding/mixing did notchange the kinetics of fiber formation, suggesting that an equilibriumbetween free peptide and fibers had been reached that prevented newnucleation and elongation reactions.

By adjusting the washing steps to the column prior to peptide elution,the concentration of the hydrophobe in the assembly buffer was modulatedprior to assembly and the requirement of the hydrophobe to promote fiberformation (FIGS. 7A-7F). Extending the washing step from 5 ml to 15 mlretained sufficient hydrophobe to stimulate fiber formation under theseconditions but washes with 30 ml of ddH₂O reduced the C18-silicaparticles that co-eluted with the peptide and yields of fiber formation.FIGS. 7A-7F show the effect of C18-column pre-wash. FIG. 7A, FIG. 7B andFIG. 7C show the effect on PilA₁₉ fiber formation with 5 ml, 15 ml or 30ml, respectively, of ddH₂O. Separate C18-columns were washed with 5 ml(standard protocol), 15 ml, and 30 ml of ddH₂O to reduce the amount ofloosely-bound C15-silica particles coeluting with PilA₁₉ during thebuffer column exchange step. After buffer exchange with a PilA₁₉solution (FIGS. 7A-7C) or a buffer-only control (FIGS. 7D-7F), theeluents were subjected to the evaporation induced assembly protocol andsamples deposited on highly oriented pyrolytic graphite (HOPG) wereimaged with an AFM operated in tapping mode.

Example 3—Biochemical and Electronic Characterization of PilA₁₉ Fibers

The optimized hydrophobe-triggered assembly protocol, with sequentialrefeeding and mixing steps, consistently produced long, flexible fibers.The average diameter of the PilA₁₉ fibers (calculated as AFM height) was˜2 nm, which is the same as that reported for the native PilA pilusfibers. AFM images of the PilA₁₉ fibers also revealed somesupramolecular structures (braids of two fibers), but most of the fiberswere present as well dispersed filaments in aqueous media (FIG. 8A).This contrasts with the extensive supramolecular aggregation of nativepili even after minimizing the pili's surface electrostatics in alkalinebuffers. The good dispersion of the PilA₁₉ fibers also permitted thecollection of CD spectra (FIG. 8B) similar to the CD profiles of otherbacteria Type IVa pili. From the CD spectrum of the PilA₁₉ fibers, anintensity ratio of 222 nm over 208 nm absorbance of 0.75 was calculated,which is close to the 0.8 intensity ratios that result from theα-helical conformation of the peptide monomers. By contrast, the CDspectra of control solutions with the native pili were convoluted bynumerous peaks (FIG. 8C) and had intensity ratios at 222 and 208 nm of1, as reported for supramolecular assemblies. This complex spectralprofile results from the random aggregation of the native pilus fibers,which form thick bundles that can only be destabilized with strongdenaturants such as urea (FIG. 8C). The aggregative nature of the nativepili is the result of surface electrostatics as well as fiber length,which can be greater and more heterogenous than in samples of ˜6-μm longPilA₁₉ fibers.

The dispersion of the PilA₁₉ fibers in aqueous media also facilitatedAFM imaging of individual filaments after deposition onto freshlycleaved highly oriented pyrolytic graphite (HOPG) (FIG. 10A). Controlsamples with the native G. sulfurreducens pili (denoted PilA fibers), onthe other hand, showed extensive supramolecular aggregation that madethe identification of single filaments more laborious (FIG. 10B).Further, the reduced aggregative nature of the PilA₁₉ fibers improvedelectrical contact with the underlying electrode. As a result,conductive probe AFM (CP-AFM) measurements of the transversal currentflowing through different fibers while sweeping the applied voltage(I-V) were less variable than with PilA fibers (FIG. 10C). Average IVcurves from four independent PilA₁₉ fibers were, however, similar tothose representative of PilA fibers (FIG. 10C-10D). Furthermore, theaverage resistance of the PilA₁₉ fibers (˜900 MOhms at ±100 mV), waswithin the orders calculated for the native wires (˜925 MOhms). I-Vcurves collected for the PilA₁₉ and PilA fibers by CP-AFM were alsosimilarly asymmetric, showing a rectification behavior such that morecurrent was measured at negative voltages than at the same positivevoltages (FIG. 10C-10D). Thus, the average rectification score(calculated as current at positive over negative voltage) for the PilA₁₉fibers was ˜0.5 and 0.7 at biological (±100 mV) and higher (±600 mV)voltages. Similarly, the PilA fibers had rectification scores below 1(˜0.7) at both. Thus, current flow through the pilus is more efficientin the electrode to the tip (more current produced at negativevoltages), which is also the biological path for the discharge ofrespiratory electrons from charged electron carriers in the cellenvelope to the pilus and then to extracellular electron acceptor.

Scanning tunneling microscopy (STM) was used to characterize nanoscalespatial variations in electronic properties within individual PilA₁₉fibers (FIG. 10E). The higher spatial resolution of the STM techniquecompared to CP-AFM resolved beadlike structural features in the PilA₁₉fibers previously described for the native PilA pili. The bright spotsare regions of the fiber with higher local electronic density of statesand, thus, regions that supply more tunneling current. The molecularsubstructures identified in the PilA₁₉ fibers have periodicities thatmatch well with those reported for the grooves and ridges that form thesurface landscape of the native pili. The STM diameter estimated for thePilA₁₉ fibers (˜5-7 nm) was also within the ranges reported for thenative PilA filaments prior to deconvoluting for the broadeningtunneling effect caused by the tip when scanning a nanowire. Also asreported previously for the PilA filaments (28), STM imaging of thePilA₁₉ fibers improved with a chemical fixation step (FIG. 10F). Thechemical treatment immobilized more fibers onto the surface and improvedelectrical contact with the electrode. As a result, the interactionsbetween the STM tip and the biomaterial were more stable when probingchemically-fixed PilA₁₉ fibers, producing cleaner STM topographic images(FIG. 10F).

Yet chemical fixation did not substantially affect the measuredconductivity, as indicated by the overlapping I-V curves collected whenprobing fixed locations of untreated (hydrated) and chemically-treatedPilA₁₉ fibers while sweeping the voltage at ±600 mV (FIG. 10G).Additionally, the STM I-V curves of untreated and treated fibersreproduced the ohmic response of the biomaterial in the ±100 mVbiological voltage range observed by CP-AFM and had similar slopes, thusa similar resistance to the passage of electrons. Plots of thedifferential conductance (dI/dV) of the untreated and treated PilA₁₉fibers versus the tip-sample bias voltage (V) confirmed thesesimilarities and revealed electronic states at low voltages that neverreach zero conductance (FIG. 10H), a distinctive electronic feature ofthe native PilA pili that results from a very small electron band gap.The STM differential conductance plots also confirmed the asymmetricconductance reported for native pili, as expected for a biomaterial thatfavors current flow from negative to positive voltages, even at the lowvoltages (i.e., ±100 mV) that drive the flow of respiratory electronsthrough the pili and onto the iron oxides (29).

DISCUSSION

The recombinant production of peptides derived from the conductive pilinof G. sulfurreducens permitted the synthesis at high yields of a solublepilin peptide (PilA₁₉) carrying an N-t truncation of the first 19 aminoacids of the mature PilA pilin. The truncated peptide lacks hydrophobicamino acids at the N-t that are known to participate in biologicalassembly processes (e.g., F1 and E5) (FIG. 1), but it retains theα-helical conformation that is needed for pilin-pilin hydrophobicinteractions and self-assembly. Importantly, the PilA₁₉ truncationpreserved the charged residues of the pilin that MD simulations predictto form salt bridges between neighboring pilins (FIG. 1), establishingintermolecular bonds critical to the structural integrity of the fibercore and the electronic coupling of aromatic side chains. Supporting thecomputational predictions, the recombinant PilA₁₉ peptide self-assembledinto a conductive fiber that exhibits biochemical, structural andelectronic properties similar to a native PilA pilus.

Octadecane, whether in solution (FIG. 9A-9B) or immobilized on silicaparticles (FIG. 5A), was a suitable hydrophobe to trigger pilinnucleation and fiber formation. The addition of the hydrophobe in asurface-constrained form also permitted its separation from the fibersby centrifugation at the end of the assembly reaction. The emission ofsilica from nanosized particles in a precise region of the UV spectrum(FIG. 6A) also proved useful to optimize the concentration ofhydrophobe. PilA₁₉ fibers were synthesized of approximately 6-μm longthat dispersed well in mild aqueous solutions (FIG. 5A-5C). Thiscontrasts with purification protocols available for native pili, whoselonger and heterogeneous length promotes the formation of largesupramolecular structures that are difficult to disrupt withoutdenaturing the pilus fiber core (FIG. 5A). The reduced aggregation ofthe PilA₁₉ fibers also facilitated their deposition on electrodesurfaces and electronic characterization by scanning probe methods (FIG.10A-10H). Reproducible electronic probing of hydrated fibers by CP-AFMis challenging, inasmuch as hydration affects the electrical contactwith the underlying electrode surface and the measured conductivity.Supramolecular aggregation in the native PilA pili enhances theseeffects and the variability of the conductivity measurements compared tothe more dispersed PilA₁₉ fibers (FIG. 10A-10H). Despite thesedifferences, scanning probe methods calculated a similar averageelectrical resistance for the PilA and PilA₁₉ fibers (900-925 MOhms) andrevealed the characteristic topographic periodicities (roughly every 10nm) that arise from the conserved helical arraignment of the pilins. Thecontact resistance was reduced between the fibers and the underlyingelectrode and, therefore, sample-to-sample variability through chemicalfixation FIG. 10A-10H Importantly, the chemical treatment did not affectthe conductive properties of the fibers, a property that can facilitatethe integration of the protein nanowires with inorganic nanomaterials inelectronic devices.

Also important for applications in bioelectronics is the rectifyingproperties of the PilA₁₉ fibers, which like the native PilA pilitransport charges more efficiently from negative to positive voltagesFIG. 10A-10H). Rectification could reflect mechanistic differences inthe directionality of charge transport through the pilus that favor thebiological flow of electrons from the cell envelope to the pilus-boundelectron acceptor.

The demonstration that truncated, conductive Type IV pilins can beexpressed in heterologous hosts, purified, and then self-assembled invitro to form fibers having an electrical conductivity comparable to thenative pili confirms unequivocally that a peptide assembly can conductelectrons in the absence of metals and/or organic redox cofactors. Theproduction of conductive nanowires also represents a significantmilestone in the field of bioelectronics in that it establishes aversatile new platform for bottom-up fabrication of nanowires thatleverages powerful, synergistic tools to customize the properties of thebiomaterial.

The production of pilin monomers and assembly into protein nanowires canbe scalable, simultaneously addressing the interrelated challenges ofsustainable supply, engineering, and production of the electronicsindustry.

Embodiments described herein provide a method of synthesizing proteinnanowires. In one embodiment, the method can comprise providing purifiedpeptide building blocks. In one embodiment, the peptide building blocksare isolated from a recombinant host. The method can include suspendingthe purified peptide building blocks in an assembly buffer and formingan assembly composition by adding a hydrophobe to the assembly buffer totrigger self-assembly of the peptide building blocks. The method caninclude increasing molecular crowding by evaporation of a volume of theassembly buffer in the assembly composition to facilitate hydrophobeguided assembly to conductive nanowires.

In one embodiment, the method can further comprise conducting one ormore elongation cycles to promote fiber formation, wherein theelongation cycle comprises providing additional peptide building blocksand/or hydrophobe into the assembly composition, mixing the assemblycomposition and evaporating the assembly buffer from the assemblycomposition.

In one embodiment, the method can comprise conducting about 4 cycles ofelongation.

In one embodiment, the hydrophobe is octadecane.

In one embodiment, the hydrophobe is added to the assembly compositionby loading the peptide building blocks into a column comprisingparticles and eluting the peptide building blocks into the assemblybuffer, wherein the elution results in the elution of a portion of theparticles of the column and the peptide building blocks into theassembly buffer and wherein the particles of the column are thehydrophobes in the assembly composition.

In one embodiment, the hydrophobe is C18-silica particles.

In one embodiment, the recombinant host is E. coli. In one embodiment,the peptide building blocks are modified PilA peptides. In oneembodiment, the peptide building blocks are truncated pilin peptidesfrom G. sulfurreducens. In one embodiment, the peptide building blocksare pilA₁₉ peptides from G. sulfurreducens. In one embodiment, thepeptide building blocks are truncated PilA_(n) peptides, wherein thetruncated PilA_(n) peptides have the amino acid sequences selected fromthe group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQID NO:5.

In one embodiment, the nanowires formed have a length from about 0.5 μmto about 10 μm. In one embodiment, the nanowires formed have a lengthfrom about 2 μm to about 8 μm. In one embodiment, the nanowires formedhave a length from about 4 μm to about 7 μm. In one embodiment, thenanowires formed have a length from about 5 μm to about 7 μm.

Embodiments described herein provide a method of synthesizing proteinnanowires. In one embodiment, the method can comprise providing purifiedpeptide building blocks, wherein the peptide building blocks areisolated from a recombinant host. The method can include suspending thepurified peptide building blocks and a hydrophobe in an assembly bufferand forming an assembly composition by loading the peptide buildingblocks into a column comprising particles and co-eluting the peptidebuilding blocks and a portion of the particles of the column into theassembly buffer and wherein the particles of the column are thehydrophobes in the assembly composition. The method can includeincreasing molecular crowding by evaporation of a volume of the assemblybuffer in the assembly composition to facilitate hydrophobe guidedassembly to conductive nanowires. In one embodiment, the hydrophobe isC18-silica particles

Embodiments described herein provide a composition comprising proteinnanowires wherein the nanowires comprise peptide building blocks derivedfrom PilA peptides and the nanowires have a length of at least about 2μM. In one embodiment, the nanowires have a length from about 2 μM toabout 10 μM. In one embodiment, the nanowires have a length from about 5μM to about 7 μM.

In one embodiment, the peptide building blocks are modified PilApeptides. In one embodiment, the peptide building blocks are truncatedPilA peptides. In one embodiment, the peptide building blocks are PilA₁₉peptides. In one embodiment, the nanowires have a length of at leastabout 2 μM. In one embodiment, the nanowires have a length from about 2μM to about 10 μM. In one embodiment, the nanowires have a length offrom about 5 μM to about 7 μM.

In one embodiment, the nanowires have an average diameter of about 2 nm.

In one embodiment, the nanowires have a Circular Dichroism (CD) profilewith an intensity ratio of 220 nm/208 nm from about 0.7 and to about 0.9or higher.

Embodiments described herein provide a device comprising synthesizedprotein nanowires wherein the nanowires comprise peptide building blocksderived from PilA peptides and the nanowires have a length of at leastabout 2 μM. In one embodiment, the nanowires have a length from about 2μM to about 10 μM. In one embodiment, the nanowires have a length offrom about 5 μM to about 7 μM.

The various embodiments described herein highlight the potential ofimproved methods functionalizing electrodes and incorporation of theimproved electrodes in BESs. The improved electrodes allow variousadditional industrial needs to be met.

All ranges given are intended to further include “any rangetherebetween” whether or not this is affirmatively stated.

All publications, patents and patent documents are incorporated byreference herein, as though individually incorporated by reference, eachin their entirety, as though individually incorporated by reference. Inthe case of any inconsistencies, the present disclosure, including anydefinitions therein, will prevail.

Although specific embodiments have been illustrated and describedherein, it will be appreciated by those of ordinary skill in the artthat any procedure that is calculated to achieve the same purpose may besubstituted for the specific embodiments shown. This application isintended to cover any adaptations or variations of the present subjectmatter.

What is claimed is:
 1. A method of synthesizing protein nanowirescomprising: providing purified peptide building blocks; suspending thepurified peptide building blocks in an assembly buffer; forming anassembly composition by addition of a hydrophobe to the assembly bufferwith the purified peptide building blocks to trigger self-assembly ofthe peptide building blocks; and increasing molecular crowding byevaporation of a volume of the assembly buffer in the assemblycomposition to facilitate hydrophobe guided assembly to conductivenanowires.
 2. The method of claim 1 further comprising conducting one ormore elongation cycles to promote fiber formation, wherein theelongation cycle comprises providing additional peptide building blocksand/or hydrophobe into the assembly composition, mixing the assemblycomposition and evaporating the assembly buffer from the assemblycomposition.
 3. The method of claim 1 wherein about four elongationcycles are conducted.
 4. The method of claim 1 wherein the hydrophobe isoctadecane.
 5. The method of claim 1 wherein the hydrophobe is added tothe assembly composition by loading the peptide building blocks into acolumn comprising particles and eluting the peptide building blocks intothe assembly buffer, wherein the elution results in the elution of aportion of the particles of the column and the peptide building blocksinto the assembly buffer and wherein the particles of the column are thehydrophobes in the assembly composition.
 6. The method of claim 5wherein the hydrophobe is C18-silica particles.
 7. The method of claim 1wherein the peptide building blocks are modified pilin peptides.
 8. Themethod of claim 1 wherein the peptide building blocks are recombinanttruncated pilin peptides from G. sulfurreducens.
 9. The method of claim1 wherein the peptide building blocks are PilA₁₉ peptides from G.sulfurreducens.
 10. The method of claim 1 wherein the nanowires formedhave a length from about 0.3 μm to about 10 μm.
 11. A compositioncomprising synthesized protein nanowires wherein the nanowires comprisepeptide building blocks derived from PilA peptides and the nanowireshave a length of at least about 2 μM.
 12. The composition of claim 11wherein the peptide building blocks are modified PilA peptides.
 13. Thecomposition of claim 11 wherein the peptide building blocks aretruncated PilA_(n) peptides, wherein the truncated PilA_(n) peptideshave the amino acid sequences selected from the group consisting of SEQID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5.
 14. The compositionof claim 11 wherein the nanowires have a length from about 2 μM to about10 μM.
 15. The composition of claim 11 wherein the nanowires have alength of from about 5 μM to about 7 μM.
 16. The composition of claim 11wherein the nanowires have an average diameter of about 2 nm.
 17. Thecomposition of claim 11 wherein the nanowires have a Circular Dichroism(CD) profile with an intensity ratio of 222 nm/208 nm from about 0.7 tohigher than
 1. 18. A device comprising synthesized protein nanowireswherein the nanowires comprise peptide building blocks derived from PilApeptides and the nanowires have a length of at least about 2 μM.
 19. Thedevice of claim 18 wherein the peptide building blocks are modified PilApeptides.
 20. The device of claim 18 wherein the device is selected fromantenna, attenuator, battery, brush, capacitor, condenser, conductor,circuit, electrode, fuel cell, generator, filter circuit breaker (fuse),inductor, coil, nanowire array, particle collector, precipitator,reactor, rectifier, relay, resistor, solar energy collector, sparkgenerator, suppressor and/or terminal.